Yeasts

Biochemistry, 1998 Sep 22, 37(38), 13379 - 91
Is a hydrophobic amino acid required to maintain the reactive V conformation of thiamin at the active center of thiamin diphosphate-requiring enzymes? Experimental and computational studies of isoleucine 415 of yeast pyruvate decarboxylase; Guo F et al.; The residue I415 in pyruvate decarboxylase from Saccharomyces cerevisiae was substituted with a variety of uncharged side chains of varying steric requirements to test the hypothesis that this residue is responsible for supporting the V coenzyme conformation reported for this enzyme {Arjunan et al . (1996) J . Mol . Biol . 256, 590-600} . Changing the isoleucine to valine and threonine decreased the kcat value and shifted the kcat-pH profile to more alkaline values progressively, indicating that the residue at position 415 not only is important for providing the optimal transition state stabilization but also ensures correct alignment of the ionizable groups participating in catalysis . Substitutions to methionine (the residue used in pyruvate oxidase for this purpose) or leucine (the corresponding residue in transketolase) led to greatly diminished kcat values, showing that for each thiamin diphosphate-dependent enzyme an optimal hydrophobic side chain evolved to occupy this key position . Computational studies were carried out on the wild-type enzyme and the I415V, I415G, and I415A variants in both the absence and the presence of pyruvate covalently bound to C2 of the thiazolium ring (the latter is a model for the decarboxylation transition state) to determine whether the size of the side chain is critically required to maintain the V conformation . Briefly, there are sufficient conformational constraints from the binding of the diphosphate side chain and three conserved hydrogen bonds to the 4'-aminopyrimidine ring to enforce the V conformation, even in the absence of a large side chain at position 415 . There appears to be increased coenzyme flexibility on substitution of Ile415 to Gly in the absence compared with the presence of bound pyruvate, suggesting that entropy contributes to the rate acceleration . The additional CH3 group in Ile compared to Val also provides increased hydrophobicity at the active center, likely contributing to the rate acceleration . The computational studies suggest that direct proton transfer to the 4'-imino nitrogen from the thiazolium C2H is eminently plausible.

Biochemistry, 1998 Sep 22, 37(38), 13276 - 84
Kinetic studies on the effect of yeast cofilin on yeast actin polymerization; Du J et al.; The effect of yeast cofilin on the kinetics of polymerization of yeast actin has been examined at 20 degrees C at both pH 8.0 and 6.6 . In the absence of cofilin, the kinetic data may be described by a simple nucleation-elongation mechanism . Kinetic data in the presence of cofilin suggests a complex dependence on the cofilin concentration . At low cofilin-to-actin ratios, cofilin increases the rate of polymerization in a way best fit by assuming filament fragmentation . The apparent fragmentation rate constants increase with increasing cofilin concentration leveling off above a cofilin-to-actin ratio of 1:8 and are independent of pH . At higher cofilin-to-actin ratios, a nonpolymerizable cofilin-G-actin complex forms resulting in a decreased rate of polymerization . The data from fluorescence photobleaching recovery experiments at low cofilin-to-actin ratios are consistent with the presence of severed filaments at both pH 8 and 6.6 . However, at pH 8 and a cofilin-to-actin ratio of 1:16, about 40-50% of the total actin is present as G-actin after polymerization while at pH 6.6 little or no G-actin is present at the same cofilin-to-actin ratio . The results suggest some cooperativity with respect to cofilin binding to filamentous actin which may be pH dependent.

Biochemistry, 1998 Sep 22, 37(38), 13091 - 101
Direct voltammetric observation of redox driven changes in axial coordination and intramolecular rearrangement of the phenylalanine-82-histidine variant of yeast iso-1-cytochrome c; Feinberg BA et al.; Direct square-wave and cyclic voltammetric electrochemical examination of the yeast iso-1-cytochrome c Phe82His/Cys102Ser variant revealed the intricacies of redox driven changes in axial coordination, concomitant with intramolecular rearrangement . Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of specific dynamic events . For the iso-1-cytochrome c Phe82His/Cys102Ser variant, square-wave voltammetry showed that the primary species in the reduced state is the Met80-Fe2+-His18 coordination form, while in the oxidized state the His82-Fe3+-His18 form predominates . The addition or removal of an electron to the appropriate form of this variant serves as a switch to a new molecular form of the cytochrome . Using the 2 x 2 electrochemical mechanism, simulations were done for the cyclic voltammetry experiments at different scan rates . These, in turn, provided relative rate constants for the intramolecular rearrangement/ligand exchange and the equilibrium redox potentials of the participating coordination forms: kb,AC = 17 s-1 for Met80-Fe3+-His18 --> His82-Fe3+-His18 and kf,BD > 10 s-1 for His82-Fe2+-His18 --> Met80-Fe2+-His18; E0' = 247 mV for Met80-Fe3+/2+-His18 couple, E0' = 47 mV for His82-Fe3+/2+-His18 couple, and E0' = 176 mV for the cross-reaction couple, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18 . Thermodynamic parameters, including the entropy of reaction, DeltaS0'Rxn, were determined for the net reduction/rearrangement reaction, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18, and compared to those for wild-type cytochrome, Met80-Fe3+-His18 + e- --> Met80-Fe2+-His18 . For the Phe82His variant mixed redox couple, DeltaS0'Rxn = -80 J/mol.K compared to DeltaS0'Rxn = -52 J/mol.K for the wild-type cyt c couple without rearrangement . Comparison of these entropies indicates that the oxidized His82-Fe3+-His18 form is highly disordered . It is proposed that this high level of disorder facilitates rapid rearrangement to Met80-Fe2+-His18 upon reduction.

J Biol Chem, 1998 Oct 2, 273(40), 25695 - 702
Cytochrome c heme lyase activity of yeast mitochondria; Tong J et al.; A highly efficient in vitro system was established for measuring by high performance liquid chromatography the formation of holocytochrome c by yeast mitochondria . Holocytochrome c formation required reducing agents, of which dithiothreitol was the most effective . With biosynthetically made, pure Drosophila melanogaster apocytochrome c and Saccharomyces cerevisiae mitochondria, the activity of cytochrome c heme lyase amounted to about 800 fmol min-1 mg-1 mitochondrial protein . The kinetics were typical Michaelis-Menten (Km approximately 1 nM), as were those of mitoplasts with broken outer membranes (Km approximately 3 nM) . As tested with mitoplasts, holocytochromes c from a range of species were found to be competitive inhibitors of heme lyase at physiological concentrations, providing a mechanism for controlling this concentration in vivo . Apocytochrome c associated with yeast mitochondria in two phases of Kd approximately 2 x 10(-10) and 10(-8) M, respectively, whereas mitoplasts had lost the high affinity binding . A site-directed mutant of apocytochrome c (lysines 5, 7, and 8 replaced by glutamine, glutamic acid, and asparagine) was found to be converted to holocytochrome c (Km approximately 3.3 nM; maximal activity unchanged), even though the mutations completely eliminated the high affinity binding . Thus, the high affinity binding of apocytochrome c to mitochondria is not directly related to holocytochrome c formation.

J Biol Chem, 1998 Oct 2, 273(40), 25637 - 46
Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants; Dick TP et al.; Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells . Using yeast 20 S proteasomes carrying different inactivated beta-subunits, we investigated the specificities and contributions of the different beta-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active beta-subunits . We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with beta5/Pre2 cleaving after hydrophobic, beta2/Pup1 after basic, and beta1/Pre3 after acidic residues, but with the exception that beta2/Pup1 and beta1/Pre3 can also cleave after some hydrophobic residues . All proteolytic activities including the "branched chain amino acid-preferring" component are associated with beta5/Pre2, beta1/Pre3, or beta2/Pup1, arguing against additional proteolytic sites . Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

J Biol Chem, 1998 Oct 2, 273(40), 25587 - 93
Import into mitochondria, folding and retrograde movement of fumarase in yeast; Knox C et al.; A single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae . All fumarase translation products are targeted and processed in mitochondria before distribution . Here we show that targeting of fumarase is coupled to translation and initially involves insertion of the protein across the mitochondrial membranes and processing by the matrix protease . Rapid folding of fumarase may determine its requirement for coupling of its translocation with translation and unique route of distribution . The amino termini of most fumarase molecules are translocated across the mitochondrial membranes and processed . Unlike the in vivo situation where these molecules are released into the cytosol, in vitro they remain externally attached to the mitochondria, thereby positioned for release from the organelle . Our model suggests that fumarase displays a unique mechanism of targeting and distribution, which occurs cotranslationally and involves folding and retrograde movement of the processed protein back through the translocation pore.

Mol Biochem Parasitol, 1998 Aug 1, 94(2), 205 - 14
Antifolate resistance due to new and known Plasmodium falciparum dihydrofolate reductase mutations expressed in yeast; Cortese JF et al.; Two new dihydrofolate reductase (DHFR) mutations were recently discovered in Plasmodium falciparum samples from an area of Bolivia with high rates of in vivo resistance to pyrimethamine-sulfadoxine: a Cys-->Arg point mutation in codon 50 and a five amino acid insertion after codon 30, termed the Bolivia repeat . We used a yeast expression system to screen these new DHFR mutants, as well as all of the other known DHFR mutant genotypes, against four antifolates: pyrimethamine, cycloguanil, chlorcycloguanil, and WR99210 . The prodrug proguanil was also evaluated . The primary 108-Asn mutation, the known secondary mutations 51-Ile, 59-Arg and 164-Leu, as well as the 50-Arg mutation, all progressively enhanced pyrimethamine resistance in naturally observed combinations with one another, with the presence of 164-Leu most significantly increasing resistance . Cycloguanil and chlorcycloguanil resistance were most impacted by 164-Leu and the paired 16-Val/108-Thr . Proguanil had no effect on malaria DHFR . All DHFRs analyzed were sensitive to WR99210 . The Bolivia repeat did not markedly affect drug sensitivity . We conclude that malaria DHFR can be reliably, rapidly and inexpensively analyzed in yeast for activity against a broad spectrum of antifolates . This system may be useful for initially characterizing newly discovered genotypes before proceeding to P . falciparum transfection; for large-scale geographic surveys of drug resistance; and for screening new antifolates or new antifolate combinations for their effectiveness against a large panel of DHFR mutants.

Mol Cell Biochem, 1998 Jul, 184(1-2), 67 - 79
Yeast mitochondrial metabolism: from in vitro to in situ quantitative study; Averet N et al.; In this work, we first compared yeast mitochondrial oxidative metabolism at different levels of organization: whole cells (C), spheroplasts (S), permeabilized spheroplasts (PS) or isolated mitochondria (M) . At present, S are more suitable for use than C for biochemical techniques such as fast extraction of metabolites and permeabilization . We show here that respiratory rates of S with various substrates are similar to C, which demonstrate that they are adapted to yeast bioenergetic studies . It appeared from ethanol metabolism +/- NAD+ or NADH respiratory rates on PS that ethanol metabolism was largely cytosolic; moreover, the activity of NADH dehydrogenase was lesser in the case of PS than in S . By comparing PS and M, the biggest difference concerned the respiratory rates of pyruvate and pyruvate-malate, which were much lower for M . Thus mitochondria preparation caused an unidentified loss involved directly in pyruvate metabolism . When the respiratory rate was lowered as a consequence of a high kinetic control of oxidative activity upstream from the respiratory chain, a similar correlation between the increase in ATP/O and decrease in respiratory rate was observed . So, the intrinsic uncoupling of proton pumps is not a particularity of M . Secondly, we demonstrate the existence of a mechanism of retarded diffusion in yeast similar to that already observed in permeabilized mammalian cells for ADP . Such a mechanism also occurs in yeast for several respiratory substrates: the K0.5 for each substrate toward the respiration rate in PS always exceeds that for M . It is proposed that such a discrepancy is due to a restriction of metabolite movement across the outer mitochondrial membrane in permeabilized cells, i.e . regulation of the substrate permeability through porin channels . In the porin-deficient yeast mutant, the K0.5 for NADH is not significantly different in either M or PS and is comparable to that of the parent strain PS . This result confirms that this retarded diffusion is essentially due to the opening-closing of the porin channel.

J Cell Biol, 1998 Sep 21, 142(6), 1501 - 17
Assembly and function of the actin cytoskeleton of yeast: relationships between cables and patches; Karpova TS et al.; Actin in eukaryotic cells is found in different pools, with filaments being organized into a variety of supramolecular assemblies . To investigate the assembly and functional relationships between different parts of the actin cytoskeleton in one cell, we studied the morphology and dynamics of cables and patches in yeast . The fine structure of actin cables and the manner in which cables disassemble support a model in which cables are composed of a number of overlapping actin filaments . No evidence for intrinsic polarity of cables was found . To investigate to what extent different parts of the actin cytoskeleton depend on each other, we looked for relationships between cables and patches . Patches and cables were often associated, and their polarized distributions were highly correlated . Therefore, patches and cables do appear to depend on each other for assembly and function . Many cell types show rearrangements of the actin cytoskeleton, which can occur via assembly or movement of actin filaments . In our studies, dramatic changes in actin polarization did not include changes in filamentous actin . In addition, the concentration of actin patches was relatively constant as cells grew . Therefore, cells do not have bursts of activity in which new parts of the actin cytoskeleton are created.

J Cell Biol, 1998 Sep 21, 142(6), 1487 - 99
A morphogenesis checkpoint monitors the actin cytoskeleton in yeast; McMillan JN et al.; A morphogenesis checkpoint in budding yeast delays cell cycle progression in response to perturbations of cell polarity that prevent bud formation (Lew, D.J., and S.I . Reed . 1995 . J . Cell Biol . 129:739- 749) . The cell cycle delay depends upon the tyrosine kinase Swe1p, which phosphorylates and inhibits the cyclin-dependent kinase Cdc28p (Sia, R.A.L., H.A . Herald, and D.J . Lew . 1996 . Mol . Biol . Cell . 7:1657- 1666) . In this report, we have investigated the nature of the defect(s) that trigger this checkpoint . A Swe1p- dependent cell cycle delay was triggered by direct perturbations of the actin cytoskeleton, even when polarity establishment functions remained intact . Furthermore, actin perturbation could trigger the checkpoint even in cells that had already formed a bud, suggesting that the checkpoint directly monitors actin organization, rather than (or in addition to) polarity establishment or bud formation . In addition, we show that the checkpoint could detect actin perturbations through most of the cell cycle . However, the ability to respond to such perturbations by delaying cell cycle progression was restricted to a narrow window of the cell cycle, delimited by the periodic accumulation of the checkpoint effector, Swe1p.

J Cell Biol, 1998 Sep 21, 142(6), 1473 - 85
A role for a protease in morphogenic responses during yeast cell fusion; Elia L et al.; Cell fusion during yeast mating provides a model for signaling-controlled changes at the cell surface . We identified the AXL1 gene in a screen for genes required for cell fusion in both mating types during mating . AXL1 is a pheromone-inducible gene required for axial bud site selection in haploid yeast and for proteolytic maturation of a-factor . Two other bud site selection genes, RSR1, encoding a small GTPase, and BUD3, were also required for efficient cell fusion . Based on double mutant analysis, AXL1 in a MATalpha strain acted genetically in the same pathway with FUS2, a fusion-dedicated gene . Electron microscopy of axl1, rsr1, and fus2 prezygotes revealed similar defects in nuclear migration, vesicle accumulation, cell wall degradation, and membrane fusion during cell fusion . The axl1 and rsr1 mutants exhibited defects in pheromone-induced morphogenesis . AXL1 protease function was required in MATalpha strains for fusion during mating . The ability of the Rsr1p GTPase to cycle was required for efficient cell fusion, as it is for bud site selection . During conjugation, vegetative functions may be redeployed under the control of pheromone signaling for mating purposes . Since Rsr1p has been reported to physically associate with Cdc24p and Bem1p components of the pheromone response pathway, we suggest that the bud site selection genes Rsr1p and Axl1p may act to mediate pheromone control of Fus2p-based fusion events during mating.

Genes Dev, 1998 Sep 15, 12(18), 2887 - 98
Repression of yeast Ste12 transcription factor by direct binding of unphosphorylated Kss1 MAPK and its regulation by the Ste7 MEK; Bardwell L et al.; The mitogen-activated protein kinase (MAPK) Kss1 has a dual role in regulating filamentous (invasive) growth of the yeast Saccharomyces cerevisiae . The stimulatory function of Kss1 requires both its catalytic activity and its activation by the MAPK/ERK kinase (MEK) Ste7; in contrast, the inhibitory function of Kss1 requires neither . This study examines the mechanism by which Kss1 inhibits invasive growth, and how Ste7 action overcomes this inhibition . We found that unphosphorylated Kss1 binds directly to the transcription factor Ste12, that this binding is necessary for Kss1-mediated repression of Ste12, and that Ste7-mediated phosphorylation of Kss1 weakens Kss1-Ste12 interaction and relieves Kss1-mediated repression . Relative to Kss1, the MAPK Fus3 binds less strongly to Ste12 and is correspondingly a weaker inhibitor of invasive growth . Analysis of Kss1 mutants indicated that the activation loop of Kss1 controls binding to Ste12 . Potent repression of a transcription factor by its physical interaction with the unactivated isoform of a protein kinase, and relief of this repression by activation of the kinase, is a novel mechanism for signal-dependent regulation of gene expression.

Fungal Genet Biol, 1998 Jun, 24(1-2), 110 - 22
The yeast cytoskeleton: the closer We look, the more We See
May KM, Hyams JS.
May, K . M., and Hyams, J . S . 1998 . The yeast cytoskeleton: The closer we look, the more we see .

FEMS Microbiol Lett, 1998 Aug 15, 165(2), 363 - 7
Fine structure of hepatitis B virus surface antigen produced by recombinant yeast: comparison with HBsAg of human origin; Yamaguchi M et al.; The ultrastructure of hepatitis B virus surface antigen (HBsAg) particles produced by recombinant yeast cells was examined using high-resolution negative staining, and ice embedding, electron microscopy . With negative staining, the HBsAg particles were spherical to slightly ovoid with a mean diameter of 27.5 nm and consisted of many subunits each 4 nm in diameter . Subunits were marked with a minute central pore . With ice embedding, particles were mostly spherical to ovoid, with a mean diameter of 23.7 nm and a 7-8 nm thick cortex surrounding an electron translucent core . Human HBsAg particles, examined using the same methods, were smaller, apparently because of molecular differences in polypeptide structure.

FEMS Microbiol Lett, 1998 Aug 15, 165(2), 335 - 40
Development of an integrative DNA transformation system for the yeast Candida utilis; Rodriguez L et al.; We report here the development of an auxotrophic transformation system for the food yeast Candida utilis . To facilitate molecular studies in Candida utilis, we isolated auxotrophic strains for uracil biosynthesis by the combination of NTG-mutagenesis and 5-fluorotic acid (FOA) selection . The ura-mutation could be functionally complemented by the homologous URA3 gene . We used both, LiAc and electroporation methods to direct insertions at the ura3 locus through homologous recombination.

J Assoc Nurses AIDS Care, 1998 Sep-Oct, 9(5), 47 - 52
Factors associated with vaginal yeast infections in HIV-positive women; Williams AB et al.; To better understand factors associated with symptomatic and asymptomatic vulvovaginal candidiasis, including the role of immune compromise and patient self-report, a cross-sectional analysis of factors associated with the isolation of yeast from vaginal swabs and clinical diagnosis of Candida vaginitis (CV) among 184 HIV-infected women was conducted . Sixty-four (35%) of the women had vaginal swabs positive for yeast . Nineteen (10%) women met the case definition for CV . In a logistic regression model, only CD4 < or = 100 cells/mm3 was predictive of CV (adds ratio = 4.5; 95% confidence interval = 1.0, 20; p = .05) . The predictive value of patient self-report of CV was only 12% . This study demonstrates that all HIV-infected women should receive a regular and thorough gynecologic evaluation, regardless of self-reported symptoms . HIV-infected women will benefit from education about prevention and treatment of CV, and women whose CD4 counts are low may wish to consider prophylaxis for CV.

Curr Biol, 1998 Aug 27, 8(17), 959 - 62
The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast; Naqvi SN et al.; Several end mutations that block the internalisation step of endocytosis in Saccharomyces cerevisiae also affect the cortical actin cytoskeleton {1} . END5 encodes a proline-rich protein (End5p or verprolin) required for a polarised cortical actin cytoskeleton and endocytosis {2,3} . End5p interacts with actin {4}, but its exact function is not yet known . To help elucidate End5p function, we sought other End5p-interacting proteins and identified the LAS17/BEE1 gene (encoding the yeast homologue of the human Wiskott-Aldrich Syndrome protein, WASp) as a high-copy-number suppressor of the temperature-sensitive growth and endocytic defects of end5-1 cells (carrying a frameshift mutation affecting the last 213 residues of End5p) . LAS17 is unable to suppress a full deletion of END5 (end5 delta), however, suggesting that the defective End5-1p in end5-1 mutants may be stabilised by Las17p . The amino terminus of Las17p interacts with the carboxyl terminus of End5p in the yeast two-hybrid system and similar interactions have been shown between WASp and a mammalian End5p homologue, WASp-interacting protein (WIP) {5} . As las17 delta deletion mutants are blocked in endocytosis, we conclude that Las17p and End5p interact and are essential for endocytosis.

Curr Biol, 1998 Sep 10, 8(18), 1031 - 4
Mutations in fission yeast Cut15, an importin alpha homolog, lead to mitotic progression without chromosome condensation; Matsusaka T et al.; Chromosome condensation is a major mitotic event . Fission yeast mutations in topoisomerase II and condensin subunits produce the characteristic 'cut' phenotypes, in which the septum bisects the nuclear material in the absence of normal condensation and sister chromatid separation . We show here that the same condensation defect is produced in cut15 temperature-sensitive mutants at the restrictive temperature (36 degrees C) . The gene product of cut15+ is, surprisingly, very similar to importin alpha, which binds proteins containing a nuclear localization signal (NLS) and forms the heterodimer with importin beta that mediates translocation through the nuclear pore complex . We show that in a nuclear import assay, purified Cut15 protein behaved identically to mammalian importin alpha but mutant Cut15 did not . Mutant Cut15 failed to bind an NLS-containing protein in vitro but could still bind importin beta . Unexpectedly, however, NLS proteins were imported into the nucleus in cut15 mutants . Cut15 is thus essential for mitotic chromosome condensation, but its role in nuclear import might be dispensable . Green fluorescent protein (GFP)-tagged Cut15 was enriched within the nucleus specifically during prometaphase-metaphase, so the interaction of Cut15 with nuclear NLS proteins during mitosis might be important for condensation.

Methods, 1998 Aug, 15(4), 315 - 21
Identification and analysis of yeast nucleosomal histone acetyltransferase complexes; Eberharter A et al.; Many studies have linked acetylation of lysine residues on the amino-terminal tails of the core histones to transcriptional activity of cellular chromatin . New insights into this field were gained on the identification of the first nuclear, type A histone acetyltransferase (HAT) . The yeast transcriptional adaptor protein Gcn5 was identified as a nuclear HAT and thus provided a direct link between pathways of transcriptional activation and histone acetylation . However, while recombinant Gcn5 can efficiently acetylate free histone H3 and, to a lesser extent, H4 it is unable to acetylate nucleosomal histones . It is therefore very likely that additional proteins are required for Gcn5-mediated acetylation of chromosomal histones . We have recently shown that Gcn5 is the catalytic subunit of two high-molecular-weight histone acetyltransferase complexes in yeast . In addition to the Gcn5-containing ADA and SAGA HAT complexes we have identified two other HAT complexes in yeast . These are called NuA3 and NuA4 for their predominant specificity to acetylate histones H3 and H4, respectively . Here we describe the identification and characterization of four native nuclear high-molecular-weight HAT complexes in Saccharomyces cerevisiae . These purified HATs can be used in a variety of functional assays to further address questions of how acetylation has an impact on transcriptional regulation.

Methods, 1998 Aug, 15(4), 295 - 302
Analyzing chromatin structure and transcription factor binding in yeast; Gregory PD et al.; The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell . This article provides two basic procedures for the study of chromatin in vivo . The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cells in vivo to determine the binding of factors to cis elements in the locus of interest . Specific examples illustrating the techniques described are given from our work on the regulation of the yeast PHO8 gene, but have also been successfully and reliably applied to the study of many other yeast loci . These procedures make it possible to correlate the binding of a transactivator with an altered or perturbed chromatin organization at a specific locus.

Electrophoresis, 1998 Aug, 19(11), 1920 - 32
Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination; Parker KC et al.; With the complete sequence of the yeast genome now available, efforts by many laboratories are underway to identify each of the spots on two-dimensional (2-D) gels corresponding to the most abundant yeast proteins . The high mass accuracy now attainable using matrix assisted laser desorption/ionization (MALDI)-mass spectrometry equipped with delayed extraction simplifies the process of identification, such that many spots can be unambiguously identified in a short period of time merely by using peptide mass fingerprinting and generally available database matching programs . Although it is not always possible to match spots between gels run by different laboratories, proteins generally yield the same abundant proteolytic fragments when tryptic digestions are performed . Databases containing these signature peptides not only simplify the task of reidentifying proteins from different gels, but also make it possible to identify small amounts of cross-contaminating proteins from different spots, as well as common extraneous contaminants such as human keratins . In this paper, we present data on the identification of > 20 previously unreported yeast proteins from 2-D gels . Some novel proteins were identified from randomly analyzed spots . Focusing on 14 spots in a narrow-pH-range gel, we demonstrate how organizing peak-table data and peptide match-list data into databases enables the identification of a larger percentage of the peaks.

J Cell Sci, 1998 Oct, 111 ( Pt 20), 3109 - 18
Yel013p (Vac8p), an armadillo repeat protein related to plakoglobin and importin alpha is associated with the yeast vacuole membrane; Fleckenstein D et al.; Proteins of the armadillo family are involved in diverse cellular processes in higher eukaryotes . Some of them, like armadillo, beta-catenin and plakoglobins have dual functions in intercellular junctions and signalling cascades . Others, belonging to the importin-alpha-subfamily are involved in NLS recognition and nuclear transport, while some members of the armadillo family have as yet unknown functions . Here, we introduce the Saccharomyces cerevisiae protein Yel013p as a novel armadillo (arm) repeat protein . The ORF Yel013w was identified in the genome project on chromosome V (EMBL: U18530) and codes for an acidic protein of 578 residues with 8 central arm-repeats, which are closely related to the central repeat-domain of Xenopus laevis plakoglobin . We show that Yel013p (Vac8p) is constitutively expressed in diploid and haploid yeasts and that it is not essential for viability and growth . However, the vacuoles of mutant cells are multilobular or even fragmented into small vesicles and the processing of aminopeptidase I, representing the cytoplasm-to-vacuole transport pathway, is strongly impaired . Consistent with these observations, subcellular fractionation experiments, immunolocalization and expression of green fluorescent protein (GFP) fusion proteins revealed that Yel013p (Vac8p) is associated with the vacuolar membrane . Our data provide evidence for the involvement of an arm-family member in vacuolar morphology and protein targeting to the vacuole.

Nature, 1998 Sep 3, 395(6697), 86 - 9
Yeast G1 cyclins are unstable in G1 phase; Schneider BL et al.; In most eukaryotes, commitment to cell division occurs in late G1 phase at an event called Start in the yeast Saccharomyces cerevisiae, and called the restriction point in mammalian cells . Start is triggered by the cyclin-dependent kinase Cdc28 and three rate-limiting activators, the G1 cyclins Cln1, Cln2 and Cln3 . Cyclin accumulation in G1 is driven in part by the cell-cycle-regulated transcription of CLN1 and CLN2, which peaks at Start . CLN transcription is modulated by physiological signals that regulate G1 progression, but it is unclear whether Cln protein stability is cell-cycle-regulated . It has been suggested that once cells pass Start, Cln proteolysis is triggered by the mitotic cyclins Clb1, 2, 3 and 4 . But here we show that G1 cyclins are unstable in G1 phase, and that Clb-Cdc28 activity is not needed fgr G1 cyclin turnover . Cln instability thus provides a means to couple Cln-Cdc28 activity to transcriptional regulation and protein synthetic rate in pre-Start G1 cells.

J Mol Biol, 1998 Sep 25, 282(3), 525 - 41
Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron; Schmidt U et al.; Previous studies show that some yeast strains carrying point mutations of domain 5 that block splicing of a mitochondrial group II intron yield spontaneous revertants in which splicing is partially restored by dominant mutations of nuclear genes . Here we cloned and sequenced the suppressor allele of one such gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F) . The MRS2 gene was first implicated in group II intron splicing by the finding that overexpression of the wild-type gene weakly suppresses the splicing defect of a mutation of another intron . Tetrad analysis showed that independently isolated suppressors of two other domain 5 mutations are also allelles of the MRS2 gene and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I and MRS2-L213M) . All three suppressor mutations cause a temperature-sensitive respiration defect that is dominant negative in heterozygous diploids, but those strains splice the mutant intron at the elevated temperature . The three mutations are in a domain of the protein that is likely to be a helix-turn-helix region, so that effects of the mutations on protein-protein interactions may contribute to these phenotypes . These mutations suppress the splicing defect of many, but not all, of the available splicing defective mutations of aI5gamma, including mutations of several intron domains . Protein and RNA blot experiments show that the level of the protein encoded by the MRS2 gene, but not the mRNA, is elevated by these mutations . Interestingly, overexpression of the wild-type protein restores much lower levels of splicing than were obtained with similar elevated levels of the mutated Mrs2 proteins . The splicing phenotypes of these strains suggest a direct role for Mrs2 protein on group II intron splicing, but an indirect effect is not yet ruled out .

J Mol Biol, 1998 Sep 25, 282(3), 505 - 23
Group II intron mobility in yeast mitochondria: target DNA-primed reverse transcription activity of aI1 and reverse splicing into DNA transposition sites in vitro; Yang J et al.; The retrohoming of the yeast mtDNA intron aI1 occurs by a target DNA-primed reverse transcription (TPRT) mechanism in which the intron RNA reverse splices directly into the recipient DNA and is then copied by the intron-encoded reverse transcriptase . Here, we carried out biochemical characterization of the intron-encoded reverse transcriptase and site-specific DNA endonuclease activities required for this process . We show that the aI1 reverse transcriptase has high TPRT activity in the presence of appropriate DNA target sites, but differs from the closely related reverse transcriptase encoded by the yeast aI2 intron in being unable to use artificial substrates efficiently . Characterization of TPRT products shows that the fully reverse spliced intron RNA is an efficient template for cDNA synthesis, while reverse transcription of partially reverse spliced intron RNA is impeded by the branch point . Novel features of the aI1 reaction include a prominent open-circular product in which cDNAs are incorporated at a nick at the antisense-strand cleavage site . The aI1 endonuclease activity, which catalyzes the DNA cleavage and reverse splicing reactions, is associated with ribonucleoprotein particles containing the intron-encoded protein and the excised intron RNA . As shown for the aI2 endonuclease, both the RNA and protein components are used for DNA target site recognition, but the aI1 protein has less stringent nucleotide sequence requirements for the reverse splicing reaction . Finally, perhaps reflecting this relaxed target specificity, in vitro experiments show that aI1 can reverse splice directly into ectopic mtDNA transposition sites, consistent with the previously suggested possibility that this mechanism is used for ectopic transposition of group II introns in vivo .

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11318 - 23
Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage; Lauer P et al.; RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site . We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs) . Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC . In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22 . By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size . Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA . The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization . In general, the genomic RARE-cleavage results support the YAC-based map . In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion . The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11241 - 5
Two yeast nuclear pore complex proteins involved in mRNA export form a cytoplasmically oriented subcomplex; Hurwitz ME et al.; We sublocalized the yeast nucleoporin Nup82 to the cytoplasmic side of the nuclear pore complex (NPC) by immunoelectron microscopy . Moreover, by in vitro binding assays we showed that Nup82 interacts with the C-terminal region of Nup159, a yeast nucleoporin that previously was also localized to the cytoplasmic side of the NPC . Hence, the two nucleoporins, Nup82 and Nup159, form a cytoplasmically oriented subcomplex that is likely to be part of the fibers emanating from the cytoplasmic ring of the NPC . Overexpression of Rss1/Gle1, a putative nucleoporin and/or mRNA transport factor, was shown previously to partially rescue depletion of Nup159 . We show here that overexpression of Rss1/Gle1 also partially rescued depletion of Nup82 . Depletion of either Nup82, Nup159, or Rss1/Gle1 was shown previously to inhibit mRNA export . As was reported previously for depletion of Nup159 or of Rss1/Gle1, we show here that depletion of Nup82 has no detectable effect on classical nuclear localization sequence-mediated nuclear import . In summary, the nucleoporins Nup159 and Nup82 form a cytoplasmically oriented subcomplex of the NPC that is likely associated with Rss1/Gle1; this complex is essential for RNA export, but not for classical nuclear localization sequence-mediated nuclear protein import.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11175 - 80
Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage; Schmidt WK et al.; Proteins terminating in the CAAX motif, for example Ras and the yeast a-factor mating pheromone, are prenylated, trimmed of their last three amino acids, and carboxyl-methylated . The enzymes that mediate these activities, collectively referred to as CAAX processing components, have been identified genetically in Saccharomyces cerevisiae . Whereas the Ram1p/Ram2p prenyltransferase is a cytosolic soluble enzyme, sequence analysis predicts that the other CAAX processing components, the Rce1p and Ste24p proteases and the Ste14p methyltransferase, contain multiple membrane spans . To determine the intracellular site(s) at which CAAX processing occurs, we have examined the localization of the CAAX proteases Rce1p and Ste24p by subcellular fractionation and indirect immunofluorescence . We find that both of these proteases are associated with the endoplasmic reticulum (ER) membrane . In addition to having a role in CAAX processing, the Ste24p protease catalyzes the first of two cleavage steps that remove the amino-terminal extension from the a-factor precursor, suggesting that the first amino-terminal processing step of a-factor maturation also occurs at the ER membrane . The ER localization of Ste24p is consistent with the presence of a carboxyl-terminal dilysine ER retrieval motif, although we find that mutation of this motif does not result in mislocalization of Ste24p . Because the ER is not the ultimate destination for a-factor or most CAAX proteins, our results imply that a mechanism must exist for the intracellular routing of CAAX proteins from the ER membrane to other cellular sites.

EMBO J, 1998 Sep 15, 17(18), 5388 - 99
Apc10 and Ste9/Srw1, two regulators of the APC-cyclosome, as well as the CDK inhibitor Rum1 are required for G1 cell-cycle arrest in fission yeast; Kominami K et al.; Many eukaryotic cells arrest the cell cycle at G1 phase upon nutrient deprivation . In fission yeast, during nitrogen starvation, cells divide twice and arrest at G1 . We have isolated a novel type of sterile mutant, which undergoes one additional S phase upon starvation and, as a result, arrests at G2 . Three loci (apc10, ste9/srw1 and rum1) were identified . The apc10 mutants, previously unidentified, show, in addition to sterility, temperature-sensitive growth with defects in chromosome segregation . apc10(+) is essential for viability, encodes a conserved protein (a homologue of budding yeast Apc10/Doc1) and is required for ubiquitination and degradation of mitotic B-type cyclins . Apc10 does not co-sediment with the 20S APC-cyclosome, a ubiquitin ligase for B-type cyclins, and in the apc10 mutant the 20S complex is intact, suggesting that it is a novel regulator for this complex . A subpopulation of Apc10 does co-immunoprecipitate with the anaphase-promoting complex (APC) . A second gene, ste9(+)/srw1(+), encodes a member of the fizzy-related family, also regulators of the APC . Finally, Rum1 is a cyclin-dependent kinase (CDK) inhibitor which exists only in G1 . The results suggest that dual downregulation of CDK, one via the APC and the other via the CDK inhibitor, is a universal mechanism that is used to arrest cell cycle progression at G1.

EMBO J, 1998 Sep 15, 17(18), 5374 - 87
Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast; Isakoff SJ et al.; Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3 . These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules . We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System) . The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast . Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC . Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity . From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1 . Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

Development, 1998 Oct, 125(20), 4055 - 66
The enhancer of polycomb gene of Drosophila encodes a chromatin protein conserved in yeast and mammals; Stankunas K et al.; The Polycomb group of genes in Drosophila are homeotic switch gene regulators that maintain homeotic gene repression through a possible chromatin regulatory mechanism . The Enhancer of Polycomb (E(Pc)) gene of Drosophila is an unusual member of the Polycomb group . Most PcG genes have homeotic phenotypes and are required for repression of homeotic loci, but mutations in E(Pc) exhibit no homeotic transformations and have only a very weak effect on expression of Abd-B . However, mutations in E(Pc) are strong enhancers of mutations in many Polycomb group genes and are also strong suppressors of position-effect variegation, suggesting that E(Pc) may have a wider role in chromatin formation or gene regulation than other Polycomb group genes . E(Pc) was cloned by transposon tagging, and encodes a novel 2023 amino acid protein with regions enriched in glutamine, alanine and asparagine . E(Pc) is expressed ubiquitously in Drosophila embryogenesis . E(Pc) is a chromatin protein, binding to polytene chromosomes at about 100 sites, including the Antennapedia but not the Bithorax complex, 29% of which are shared with Polycomb-binding sites . Surprisingly, E(Pc) was not detected in the heterochromatic chromocenter . This result suggests that E(Pc) has a functional rather than structural role in heterochromatin formation and argues against the heterochromatin model for PcG function . Using homology cloning techniques, we identified a mouse homologue of E(Pc), termed Epc1, a yeast protein that we name EPL1, and as well as additional ESTs from Caenorhabditis elegans, mice and humans . Epc1 shares a long, highly conserved domain in its amino terminus with E(Pc) that is also conserved in yeast, C . elegans and humans . The occurrence of E(Pc) across such divergent species is unusual for both PcG proteins and for suppressors of position-effect variegation, and suggests that E(Pc) has an important role in the regulation of chromatin structure in eukaryotes.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 1025 - 30
Bullera penniseticola sp . nov . and Kockovaella sacchari sp . nov., two new yeast species isolated from plants in Thailand; Takashima M et al.; Two strains of ballistocondium-forming yeasts, isolated from plants collected in the south-east seacoast of Bangkok, Thailand, were described . The strains (K-272(T) and K-337(T)) were assigned to the genera Bullera and Kockovaella, respectively, based on morphological and chemotaxonomical characteristics . Phylogenetically, strain K-272(T) is close to Bullera hannae, and strain K-337(T) is close to Kockovaella thailandica and Kockovaella imperatae . These two strains represent new species based on DNA-DNA reassociation experiments . Bullera penniseticola Takashima et Nakase sp . nov . and Kockovaella sacchari Takashima et Nakase sp . nov . are proposed for K-272(T) (=JCM 9857(T)) and K-337(T) (=JCM 9858(T)), respectively.

Biochim Biophys Acta, 1998 Sep 2, 1373(2), 321 - 31
Cloning of a cDNA coding for an amino acid carrier from Ricinus communis (RcAAP1) by functional complementation in yeast: kinetic analysis, inhibitor sensitivity and substrate specificity; Marvier AC et al.; A cDNA for the amino acid permease gene RcAAP1 has been isolated from Ricinus communis by yeast complementation and subjected to a detailed kinetic analysis . RcAAP1 cDNA is 1.5 kb with an open reading frame that codes for a protein with 486 amino acids and a calculated molecular mass of 53.1 kDa . RcAAP1-mediated histidine uptake was pH dependent with highest transport rates at acidic pH; it was sensitive to protonophores and uncouplers and the Km for histidine uptake was 96 microM . The substrate specificity was investigated by measuring the levels of inhibition of histidine uptake by a range of amino acids . The basic amino acids (histidine, lysine and arginine) showed strongest inhibition of uptake whereas acidic amino acids competed less effectively . Alanine was the most efficient competitor of the neutral amino acids . Glutamine, serine, asparagine, methionine and cysteine showed moderate inhibition whereas threonine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan showed only low levels of inhibition . Glycine, proline and citrulline caused slight stimulation . More detailed competition kinetics indicated that both lysine and arginine showed simple competitive inhibition of histidine uptake . When direct uptake measurements were carried out, both lysine and arginine were found to be effective substrates for RcAAP1.

J Biol Chem, 1998 Sep 18, 273(38), 24963 - 71
Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution; Harley CA et al.; The determinants of transmembrane protein insertion orientation at the endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using variants of a Type III (naturally exofacial N terminus (Nexo)) transmembrane fusion protein derived from the N terminus of Ste2p, the alpha-factor receptor . Small positive and negative charges adjacent to the transmembrane segment had equal and opposite effects on orientation, and this effect was independent of N- or C-terminal location, consistent with a purely electrostatic interaction with response mechanisms . A 3:1 bias toward Nexo insertion, observed in the absence of a charge difference, was shown to reflect the Nexo bias conferred by longer transmembrane segments . Orientation correlated best with total hydrophobicity rather than length, but it was also strongly affected by the distribution of hydrophobicity within the transmembrane segment . The most hydrophobic terminus was preferentially translocated . Insertion orientation thus depends on integration of responses to at least three parameters: charge difference across a transmembrane segment, its total hydrophobicity, and its hydrophobicity gradient . Relative signal strengths were estimated, and consequences for topology prediction are discussed . Responses to transmembrane sequence may depend on protein-translocon interactions, but responses to charge difference may be mediated by the electrostatic field provided by anionic phospholipids.

J Bioenerg Biomembr, 1998 Jun, 30(3), 245 - 55
Binding of rat brain hexokinase to recombinant yeast mitochondria: effect of environmental factors and the source of porin; Aflalo C et al.; Heterologous binding of rat brain hexokinase to wild type, porinless, and recombinant yeast mitochondria expressing human porin was assessed, partially characterized, and compared to that in the homologous system (rat liver mitochondria) . With porin-containing yeast mitochondria it is shown that (i) a significant, saturable association occurs; (ii) its extent and apparent affinity, correlated with the origin of porin, are enhanced in the presence of dextran; (iii) the binding requires Mg ions and apparently follows a complex cooperative mechanism . This heterologous association does not seem to differ fundamentally from that in the homologous system and represents a good basis for molecular studies in yeast . With porinless yeast mitochondria, binding occurs at much lower affinity, but to many more sites per mitochondrion . The results indicating a major but not exclusive role for porin in the binding are discussed in terms of (i) the mode and mechanism of binding, and (ii) the suitability of the rat hexokinase-yeast mitochondria couple for the study of heterogeneous catalysis in reconstituted cellular model systems.

J Cell Biol, 1998 Sep 7, 142(5), 1289 - 99
The yeast V159N actin mutant reveals roles for actin dynamics in vivo; Belmont LD et al.; Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release . Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover . Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast . Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity . However, fluid phase endocytosis is defective in act1-159 strains . act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle . In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins . Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.

Genes Dev, 1998 Sep 1, 12(17), 2684 - 97
Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gbetagamma complex underlies activation of the yeast pheromone response pathway; Pryciak PM et al.; In the Saccharomyces cerevisiae pheromone response pathway, the Gbetagamma complex activates downstream responses by an unknown mechanism involving a MAP kinase cascade, the PAK-like kinase Ste20, and a Rho family GTPase, Cdc42 . Here we show that Gbetagamma must remain membrane-associated after release from Galpha to activate the downstream pathway . We also show that pheromone stimulates translocation of the kinase cascade scaffold protein Ste5 to the cell surface . This recruitment requires Gbetagamma function and the Gbetagamma-binding domain of Ste5, but not the kinases downstream of Gbetagamma, suggesting that it is mediated by Gbetagamma itself . Furthermore, this event has functional significance, as artificial targeting of Ste5 to the plasma membrane, but not intracellular membranes, activates the pathway in the absence of pheromone or Gbetagamma . Remarkably, although independent of Gbetagamma, activation by membrane-targeted Ste5 requires Ste20, Cdc42, and Cdc24, indicating that their participation in this pathway does not require them to be activated by Gbetagamma . Thus, membrane recruitment of Ste5 defines a molecular activity for Gbetagamma . Moreover, our results suggest that this event promotes kinase cascade activation by delivering the Ste5-associated kinases to the cell surface kinase Ste20, whose function may depend on Cdc42 and Cdc24.

Yeast, 1998 Aug, 14(11), 1027 - 39
Functional analysis of three adjacent open reading frames from the right arm of yeast chromosome XVI; Waskiewicz-Staniorowska B et al.; A 7.24 kb genomic DNA fragment from the yeast Saccharomyces cerevisiae chromosome XVI was isolated by complementation of a new temperature-sensitive mutation tsa1 . We determined the nucleotide sequence of this fragment located on the right arm of chromosome XVI . Among the three, complete open reading frames: YPR041w, YPR042c and YPR043w contained within this fragment, the gene YPR041w was shown to complement the tsa1 mutation and to correspond to the TIF5 gene encoding an essential protein synthesis initiation translation factor . The YPR042c gene encodes a hypothetical protein of 1075 amino acids containing four putative transmembrane segments and is non-essential for growth . The gene YPR043c encoding the 10 kDa product, highly similar to the human protein L37a from the 60S ribosomal subunit, was found to be essential and a dominant lethal . We conclude that three tightly linked yeast genes are involved in the translation process.

Yeast, 1998 Aug, 14(11), 985 - 1000
Multiple regulatory proteins mediate repression and activation by interaction with the yeast Mig1 binding site; Wu J et al.; A major mediator of glucose repression in yeast is Mig1, a zinc finger protein that binds to a GC-rich recognition sequence found upstream of many glucose-repressible genes . Because these Mig1 sites are found upstream of genes under different modes of regulation, we studied regulation of transcription mediated by an isolated Mig1 site placed upstream of a reporter gene under control of UAS(CYC1) . The Mig1 site responded appropriately to glucose control and regulatory mutations, including snf1, reg1, cyc8, and tup1, mimicking the behavior of the SUC2 gene . Deletion of the MIG1-coding gene reduced but did not eliminate glucose repression mediated by the Mig1 site . Complete loss of repression was seen in a mig1 mig2 double mutant . When the UAS(CYC1) was replaced by UAS(ADH1) in the reporter plasmid, the Mig1 site activated transcription under most conditions . Mutations of the two Mig1 binding sites in the SUC2 promoter resulted in loss of activation of SUC2 expression . These results suggest the presence of an unknown activator or activators that binds to the Mig1 site . The activator is not any of the proteins previously proposed to bind to this site, including Mig1, Mig2, Msn2, or Msn4 . Band shift assays showed that Mig1 is the major protein in yeast cell extracts that binds to the Mig1 site in vitro . This binding is not regulated by glucose or mutations in CYC8 or TUP1.

Biochem J, 1998 Sep 15, 334 ( Pt 3), 723 - 9
ATP-dependent transport of reduced glutathione in yeast secretory vesicles; Rebbeor JF et al.; Turnover of cellular reduced glutathione (GSH) is accomplished predominantly by export into the extracellular space; however, the plasma membrane transport mechanisms that mediate GSH efflux are not well characterized . The present study examined GSH transport using secretory vesicles isolated from the sec6-4 mutant strain of Saccharomyces cerevisiae . In contrast with studies in mammalian membrane vesicles, GSH transport in yeast secretory vesicles was mediated largely by an ATP-dependent, low-affinity pathway (Km 19+/-5 mM) . ATP-dependent {3H}GSH transport was cis-inhibited by substrates of the yeast YCF1 transporter, including sulphobromophthalein, glutathione S-conjugates and the alkaloid verapamil, and was competitively inhibited by S-(2, 4-dinitrophenyl)glutathione (DNP-SG) . Similarly, GSH competitively inhibited ATP-dependent {3H}DNP-SG transport, with a Ki of 18+/-2 mM, but had no effect on ATP-dependent {3H}taurocholate transport . ATP-dependent GSH transport was not affected by either membrane potential or pH-gradient uncouplers, but was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonate, probenecid and sulphinpyrazone, which are inhibitors of mrp1 and mrp2, mammalian homologues of the yeast YCF1 transporter . Western blot analysis of the secretory vesicle membrane fraction confirmed the presence of Ycf1p . These results provide the first direct evidence for low-affinity, ATP-dependent transport of GSH, and demonstrate that this ATP-dependent pathway displays kinetic characteristics similar to those of the yeast YCF1 transporter.

J Biol Chem, 1998 Sep 11, 273(37), 23704 - 8
Bcl-xS and Bad potentiate the death suppressing activities of Bcl-xL, Bcl-2, and A1 in yeast; Tao W et al.; Members of the Bcl-2 family can be grouped into three classes based upon their effects on cell death . The first class suppresses death and includes Bcl-2 . A second group, which includes Bax, is lethal, whereas a third class, including Bcl-xS, potentiates killing, although the members are not lethal by themselves . The proteins in the last class are proposed to exert their activity by binding to anti-apoptotic family members, thereby making the cell more susceptible to killing by another agent . To test this hypothesis, an inducible yeast expression system is reported that permits the functional analysis of three Bcl-2 family members . In yeast, Bax is lethal, and this activity is suppressed by Bcl-xL, Bcl-2, and A1 . Co-expression of Bcl-xS did not diminish the ability of any of the anti-apoptotic members to antagonize Bax . Rather, co-expression of Bcl-xS potentiated the anti-death activity of all three proteins . This effect was not the result of changes in either the levels or integrity of Bax or anti-apoptotic proteins . Thus, Bcl-xS can bind to anti-apoptotic family members, but this association does not result in loss of biological activity . Therefore, Bcl-xS may act downstream of Bax and in a pathway that is conserved in yeast.

J Biol Chem, 1998 Sep 11, 273(37), 23690 - 7
The role of 3'-5' exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance; Vanderstraeten S et al.; In the D171G/D230A mutant generated at conserved aspartate residues in the Exo1 and Exo2 sites of the 3'-5' exonuclease domain of the yeast mitochondrial DNA (mtDNA) polymerase (pol-gamma), the mitochondrial genome is unstable and the frequency of mtDNA point mutations is 1500 times higher than in the wild-type strain and 10 times higher than in single substitution mutants . The 10(4)-fold decrease in the 3'-5' exonuclease activity of the purified mtDNA polymerase is associated with mismatch extension and high rates of base misincorporation . Processivity of the purified polymerase on primed single-stranded DNA is decreased and the Km for dNTP is increased . The sequencing of mtDNA point mutations in the wild-type strain and in proofreading and mismatch-repair deficient mutants shows that mismatch repair contributes to elimination of the transitions while exonucleolytic proofreading preferentially repairs transversions, and more specifically A to T (or T to A) transversions . However, even in the wild-type strain, A to T (or T to A) transversions are the most frequent substitutions, suggesting that they are imperfectly repaired . The combination of both mismatch repair and proofreading deficiencies elicits a mitochondrial error catastrophe . These data show that the faithful replication of yeast mtDNA requires both exonucleolytic proofreading and mismatch repair.

J Biol Chem, 1998 Sep 11, 273(37), 23641 - 4
Subunits of the yeast SWI/SNF complex are members of the actin-related protein (ARP) family; Peterson CL et al.; The yeast SWI/SNF chromatin remodeling complex is comprised of 11 tightly associated polypeptides (SWI1, SWI2, SWI3, SNF5, SNF6, SNF11, SWP82, SWP73, SWP59, SWP61, and SWP29) . We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify the genes that encode the SWP59 and SWP61 subunits . Surprisingly, we find that SWP59 and SWP61 are encoded by the ARP9 and ARP7 genes, respectively, which encode members of the actin-related protein (ARP) family . Sequence analyses have shown that ARP9 and ARP7 are 24-26% identical (48-51% similar) to yeast actin and that they are likely to maintain the overall actin fold . Deletion of either the ARP9 or ARP7 gene causes typical swi/snf phenotypes, including growth defects on media containing galactose, glycerol, or sucrose as sole carbon sources . ARP9 and ARP7 are also required for expression of an HO-lacZ fusion gene and for full transcriptional enhancement by the GAL4 activator . The identification of two ARP family members as crucial subunits of the SWI/SNF complex suggests that the complex may contain a total of three different ATPase subunits; furthermore, the similarity of ARP7 and ARP9 to the HSP and HSC family of ATPases suggests the possibility that chromatin remodeling by SWI/SNF may involve chaperone-like activities.

J Environ Pathol Toxicol Oncol, 1998, 17(3-4), 305 - 11
Effect of selenium-enriched yeast pretreatment on the antioxidative defense in the skin of rats exposed to heat shock; Korac B et al.; Skin protection against heat shock and the specificity in the organization of antioxidative defenses were examined in rats given oral antioxidative pretreatment with selenium (Se)-enriched yeast and vitamins E, C, and A for 15 days and then exposed to hyperthermia . The activity of antioxidative enzymes in the skin and the liver was monitored 1 hour and 3 hours after heat shock . Glutathione peroxidase (GSH-Px) activity was increased in the skin after heat shock in the groups supplemented with antioxidants, but not in the controls . In contrast, the activity of liver GSH-Px was increased only in the controls receiving antioxidants . Heat shock led to a decrease in liver superoxide dismutase (SOD) activity at 1 hour in the antioxidant-supplemented group, but this was unchanged in the liver of all other groups and in the skin . The activity of thioredoxin reductase (TR) in the skin was increased in the antioxidant supplemented group 1 hour after heat shock, whereas the hepatic thioredoxin reductase activity was decreased . The activities of catalase (CAT), glutathione reductase (GR), and glutathione-S-transferase (GST) were unaffected by either treatment . These results suggest that supplementation with antioxidants protects the skin against heat shock, especially with respect to the GSH-Px and TR activity . The different response of the skin in comparison with the liver probably reflects differences in organization and regulation of antioxidative defenses.

Mol Biol Cell, 1998 Sep, 9(9), 2667 - 80
The dynamics of golgi protein traffic visualized in living yeast cells; Wooding S et al.; We describe for the first time the visualization of Golgi membranes in living yeast cells, using green fluorescent protein (GFP) chimeras . Late and early Golgi markers are present in distinct sets of scattered, moving cisternae . The immediate effects of temperature-sensitive mutations on the distribution of these markers give clues to the transport processes occurring . We show that the late Golgi marker GFP-Sft2p and the glycosyltransferases, Anp1p and Mnn1p, disperse into vesicle-like structures within minutes of a temperature shift in sec18, sft1, and sed5 cells, but not in sec14 cells . This is consistent with retrograde vesicular traffic, mediated by the vesicle SNARE Sft1p, to early cisternae containing the target SNARE Sed5p . Strikingly, Sed5p itself moves rapidly to the endoplasmic reticulum (ER) in sec12 cells, implying that it cycles through the ER . Electron microscopy shows that Golgi membranes vesiculate in sec18 cells within 10 min of a temperature shift . These results emphasize the dynamic nature of Golgi cisternae and satisfy the kinetic requirements of a cisternal maturation model in which all resident proteins must undergo retrograde vesicular transport, either within the Golgi complex or from there to the ER, as anterograde cargo advances.

Mol Biol Cell, 1998 Sep, 9(9), 2611 - 26
Sequence determinants for regulated degradation of yeast 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein; Gardner R et al.; The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway . To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p . When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized . The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation . When amino acid residues 27-54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated . The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes . These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes . Neither determinant was sufficient to impart regulated degradation to Hmg1p . These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.

Mol Biol Cell, 1998 Sep, 9(9), 2545 - 60
Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities; Kaldis P et al.; Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks) . Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK) . Human CAK has been identified as a p40(MO15)/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II . In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity . Here we report that Cak1p and p40(MO15) have strikingly different substrate specificities . Cak1p preferentially phosphorylated monomeric cdks, whereas p40(MO15) preferentially phosphorylated cdk/cyclin complexes . Furthermore, p40(MO15) only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3 . We also found that cdk inhibitors, including p21(CIP1), p27(KIP1), p57(KIP2), p16(INK4a), and p18(INK4c), could block phosphorylation by p40(MO15) but not phosphorylation by Cak1p . Our results demonstrate that although both Cak1p and p40(MO15) activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly . Structural and physiological implications of these findings will be discussed.

Genetics, 1998 Sep, 150(1), 43 - 58
Identification of functional connections between calmodulin and the yeast actin cytoskeleton; Sekiya-Kawasaki M et al.; One of four intragenic complementing groups of temperature-sensitive yeast calmodulin mutations, cmd1A, results in a characteristic functional defect in actin organization . We report here that among the complementing mutations, a representative cmd1A mutation (cmd1-226: F92A) is synthetically lethal with a mutation in MYO2 that encodes a class V unconventional myosin with calmodulin-binding domains . Gel overlay assay shows that a mutant calmodulin with the F92A alteration has severely reduced binding affinity to a GST-Myo2p fusion protein . Random replacement and site-directed mutagenesis at position 92 of calmodulin indicate that hydrophobic and aromatic residues are allowed at this position, suggesting an importance of hydrophobic interaction between calmodulin and Myo2p . To analyze other components involved in actin organization through calmodulin, we isolated and characterized mutations that show synthetic lethal interaction with cmd1-226; these "cax" mutants fell into five complementation groups . Interestingly, all the mutations themselves affect actin organization . Unlike cax2, cax3, cax4, and cax5 mutations, cax1 shows allele-specific synthetic lethality with the cmd1A allele . CAX1 is identical to ANP1/GEM3/MCD2, which is involved in protein glycosylation . CAX4 is identical to the ORF YGR036c, and CAX5 is identical to MNN10/SLC2/BED1 . We discuss possible roles for Cax proteins in the regulation of the actin cytoskeleton.

Genetics, 1998 Sep, 150(1), 21 - 30
The {KIL-d} cytoplasmic genetic element of yeast results in epigenetic regulation of viral M double-stranded RNA gene expression; Talloczy Z et al.; {KIL-d} is a cytoplasmically inherited genetic trait that causes killer virus-infected cells of Saccharomyces cerevisiae to express the normal killer phenotypes in a/alpha cells, but to show variegated defective killer phenotypes in a or alpha type cells . Mating of {KIL-d} haploids results in "healing" of their phenotypic defects, while meiosis of the resulting diploids results in "resetting" of the variegated, but mitotically stable, defects . We show that {KIL-d} does not reside on the double-stranded RNA genome of killer virus . Thus, the {KIL-d} effect on viral gene expression is epigenetic in nature . Resetting requires nuclear events of meiosis, since {KIL-d} can be cytoplasmically transmitted during cytoduction without causing defects in killer virus expression . Subsequently, mating of these cytoductants followed by meiosis generates spore clones expressing variegated defective phenotypes . Cytoduction of wild-type cytoplasm into a phenotypically defective {KIL-d} haploid fails to heal, nor does simultaneous or sequential expression of both MAT alleles cause healing . Thus, healing is not triggered by the appearance of heterozygosity at the MAT locus, but rather requires the nuclear fusion events which occur during mating . Therefore, {KIL-d} appears to interact with the nucleus in order to exert its effects on gene expression by the killer virus RNA genome.

Genetics, 1998 Sep, 150(1), 11 - 9
Characterization of an allele-nonspecific intragenic suppressor in the yeast plasma membrane H+-ATPase gene (Pma1); Maldonado AM et al.; We have analyzed the ability of A165V, V169I/D170N, and P536L mutations to suppress pma1 dominant lethal alleles and found that the P536L mutation is able to suppress the dominant lethality of the pma1-R271T, -D378N, -D378E, and -K474R mutant alleles . Genetic and biochemical analyses of site-directed mutants at Pro-536 suggest that this amino acid may not be essential for function but is important for biogenesis of the ATPase . Proteins encoded by dominant lethal pma1 alleles are retained in the endoplasmic reticulum, thus interfering with transport of wild-type Pma1 . Immunofluorescence studies of yeast conditionally expressing revertant alleles show that the mutant enzymes are correctly located at the plasma membrane and do not disturb targeting of the wild-type enzyme . We propose that changes in Pro-536 may influence the folding of the protein encoded by a dominant negative allele so that it is no longer recognized and retained as a misfolded protein by the endoplasmic reticulum.

Trends Genet, 1998 Aug, 14(8), 317 - 21
A locus control region regulates yeast recombination; Haber JE; The yeast Saccharomyces can switch its mating type by a highly choreographed recombination event in which 'a' or 'alpha' sequences at the mating-type (MAT) locus are replaced by opposite mating-type sequences copied from one of two donors, HML and HMR, located near the two ends of the same chromosome III . MAT alpha cells 'know' to choose HML, while MAT alpha cells preferentially recombine with HMR . Donor preference is regulated by a 250 bp recombination enhancer, that controls recombination of the entire left arm of chromosome III . Recent studies have shown how this locus-control region is turned on and off.

Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10584 - 9
The ATPase and protease domains of yeast mitochondrial Lon: roles in proteolysis and respiration-dependent growth; van Dijl JM et al.; The ATP-dependent Lon protease of Saccharomyces cerevisiae mitochondria is required for selective proteolysis in the matrix, maintenance of mitochondrial DNA, and respiration-dependent growth . Lon may also possess a chaperone-like function that facilitates protein degradation and protein-complex assembly . To understand the influence of Lon's ATPase and protease activities on these functions, we examined several Lon mutants for their ability to complement defects of Lon-deleted yeast cells . We also developed a rapid procedure for purifying yeast Lon to homogeneity to study the enzyme's activities and oligomeric state . A point mutation in either the ATPase or the protease site strongly inhibited the corresponding activity of the pure protein but did not alter the protein's oligomerization; when expressed at normal low levels neither of these mutant enzymes supported respiration-dependent growth of Lon-deleted cells . When the ATPase- or the protease-containing regions of Lon were expressed as separate truncated proteins, neither could support respiration-dependent growth of Lon-deleted cells; however, coexpression of these two separated regions sustained wild-type growth . These results suggest that yeast Lon contains two catalytic domains that can interact with one another even as separate proteins, and that both are essential for the different functions of Lon.

EMBO J, 1998 Sep 1, 17(17), 5026 - 36
Cell-cycle arrest and inhibition of G1 cyclin translation by iron in AFT1-1(up) yeast; Philpott CC et al.; Although iron is an essential nutrient, it is also a potent cellular toxin, and the acquisition of iron is a highly regulated process in eukaryotes . In yeast, iron uptake is homeostatically regulated by the transcription factor encoded by AFT1 . Expression of AFT1-1(up), a dominant mutant allele, results in inappropriately high rates of iron uptake, and AFT1-1(up) mutants grow slowly in the presence of high concentrations of iron . We present evidence that when Aft1-1(up) mutants are exposed to iron, they arrest the cell division cycle at the G1 regulatory point Start . This arrest is dependent on high-affinity iron uptake and does not require the activation of the DNA damage checkpoint governed by RAD9 . The iron-induced arrest is bypassed by overexpression of a mutant G1 cyclin, cln3-2, and expression of the G1-specific cyclins Cln1 and Cln2 is reduced when yeast are exposed to increasing amounts of iron, which may account for the arrest . This reduction is not due to changes in transcription of CLN1 or CLN2, nor is it due to accelerated degradation of the protein . Instead, this reduction occurs at the level of Cln2 translation, a recently recognized locus of cell-cycle control in yeast.

Int J Biochem Cell Biol, 1998 Jul, 30(7), 783 - 96
Characterization of an exocellular protein phosphatase with dual substrate specificity from the yeast Yarrowia lipolytica; Jolivet P et al.; In previous work, the major endocellular protein phosphatase activity has been identified in the secretory yeast Yarrowia lipolytica as a PP2A . The aim of the present work was to seek the presence of one protein phosphatase excreted in the exocellular medium and to study its activity during yeast growth in media supplemented or not supplemented with inorganic phosphate . Protein phosphatase was purified and activity was assayed by following the dephosphorylation of three substrates, {32P}casein, phosphotyrosine and a synthetic tyrosine-phosphorylated peptide . Phosphatase activity recovered in the medium after 25 h culture was greatly enhanced by Pi-deficiency . After several purification steps, the enzyme preparation presents an apparent electrophoretic homogeneity on SDS-PAGE with associated phosphoseryl/threonyl and phosphotyrosyl activities . The kinetic properties exclude contamination by a copurified protein and it is concluded that the two activities are carried by the same single proteic species . It was characterized by gel filtration as a 33 kDa protein with one single subunit demonstrated by SDS-PAGE . An absolute requirement for reducing-agents is observed suggesting that the enzyme contains at least one essential reactive cysteinyl residue . Optimum pH value is 6.1, apparent K(m) for phosphotyrosine was calculated to be 760 microM and Hill coefficient 3.2 indicating a rather high cooperativity . These results showed that the involvement of alkaline and/or acid phosphatase was unlikely . In conclusion, a protein phosphatase distinct from endocellular PP2A is secreted by Yarrowia lipolytica and characterized as a phosphotyrosine protein phosphatase with associated phosphoseryl/threonyl activity.

Nucleic Acids Res, 1998 Sep 15, 26(18), 4186 - 95
Biochemical and genetic characterization of the dominant positive element driving transcription ofthe yeast TBP-encoding gene, SPT15; Schroeder SC et al.; We previously demonstrated that a combination of both positive and negative cis -acting upstream elements control the transcription of the gene encoding TBP ( SPT15 ) in Saccharomyces cerevisiae . One of these elements found in that study, resident between 5' flanking sequences -147 and -128 , and termed PED (for positive element distal), was found to play an essential positive role in driving transcription of the gene encoding TBP . In this report, we map at nucleotide-level resolution, the critical residues which comprise PED, purify and sequence the protein that binds to it and determine that this PED binding factor is Abf1p, an abundant yeast protein previously broadly implicated in both gene regulation and DNA replication . In the case of the TBP-encoding gene, however, Abf1p works through the PED element which is a non-consensus binding site . Based upon the work of others, the PED-variant ABF1 site would be predicted to be a very poor binding site for this factor yet Abf1p binds PED and a consensus ABF1 site with comparable affinity . These results are discussed in light of the broader context of Abf1p-mediated gene regulation.

J Cell Biol, 1998 Aug 24, 142(4), 949 - 61
A large PEST-like sequence directs the ubiquitination, endocytosis, and vacuolar degradation of the yeast a-factor receptor; Roth AF et al.; The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode . Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G . Davis . 1996 . J . Cell Biol . 134:661-674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD) . Here, we concentrate on the Ste3p sequences required for constitutive endocytosis . Constitutive endocytosis is rapid . Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t1/2 of 15 min . Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover . Deletions intruding into this interval block or severely slow the rate of endocytic turnover . Moreover, the same 36-residue sequence directs receptor ubiquitination . Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination . Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient . When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation . Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity-no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval . The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences-a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins . A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal . Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p . Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.

Mol Microbiol, 1998 Jul, 29(2), 515 - 26
Overexpression of MID2 suppresses the profilin-deficient phenotype of yeast cells; Marcoux N et al.; Profilin-deficient Saccharomyces cerevisiae cells show abnormal growth, actin localization, chitin deposition, bud formation and cytokinesis . Previous studies have also revealed a synthetic lethality between pfy1 and late secretory mutants, suggesting a role for profilin in intracellular transport . In this work, we document further the secretion defect associated with the pfy1delta mutant . Electron microscopic observations reveal an accumulation of glycoproteins in the bud and in the mother cell . The MATa, pfy1delta cells mate as well as wild-type cells, while the mating efficiency of MAT alpha, pfy1delta cells is reduced . Pulse-chase experiments demonstrate an accumulation of the 19 kDa alpha-factor precursor and delayed secretion of the mature alpha-factor . The TGN protein Kex2p is the principal enzyme responsible for the endoproteolytic cleavage of the alpha-factor precursor . An immunofluorescence detection of Kex2p shows an altered localization in pfy1delta cells . Instead of a discrete punctate distribution, the enzyme is dispersed throughout the cytoplasm . A high-copy-number plasmid containing MID2, which encodes a potential transmembrane protein involved in cell cycle control, suppresses the abnormal growth, actin distribution, alpha-factor maturation and the accumulation of intracellular membranous structures in pfy1delta cells.

Chem Phys Lipids, 1998 Jun, 93(1-2), 47 - 55
Characterization of the Candida rugosa lipase system and overexpression of the lip1 isoenzyme in a non-conventional yeast; Mileto D et al.; The fungus C . rugosa produces lipase isoenzymes (CRLs) homologous to the Geotrichum candidum and Yarrowia lipolytica lipases to which they share ca . 40 and 30% sequence identity, with a domain of sequence conservation at the N-terminal half of the protein . CRL proteins have high sequence homology but are not identical in their catalytic activity, therefore calling for the resolution of isoforms via heterologous expression . The non-conventional use of a serine codon in several Candida species frustrates overexpression in the currently available host systems . The LIP1 gene, coding for the major CRL form, was therefore expressed in C . maltosa, a related fungus with the same codon usage as C . rugosa . A recombinant lipase was produced and secreted in an active form in the culture medium upon engineering the 5' and 3' ends of the gene.

Biosci Biotechnol Biochem, 1998 Jul, 62(7), 1332 - 6
Anomer-selective glucosylation of l-menthol by yeast alpha-glucosidase; Nakagawa H et al.; l-Menthol was glucosylated by the alpha-glucosidase (EC 3.2.1.20) of Saccharomyces cerevisiae using maltose as the glucosyl donor . When 50 mg of l-menthol and 1.6 M maltose in 10 mM citrate-phosphate buffer (pH 5.5) were incubated at 45 degrees C, l-menthyl alpha-D-glucopyranoside (alpha-MenG) was alpha-anomer-selectively formed as a product . The specificity of the alpha-linkage was confirmed by 13C-NMR analysis . In the reaction mixture after 2 h, alpha-MenG was mainly accumulated in a crystalline form and the concentration of dissolved alpha-MenG was constant at 1.4 mM . The molar conversion yield of alpha-MenG produced based on the supplied l-menthol was maximally 30.7% at 48 h of reaction.

Anim Genet, 1998 Jun, 29(3), 216 - 9
Construction of a bovine yeast artificial chromosome (YAC) library; Takeda H et al.; We have constructed a bovine yeast artificial chromosome (YAC) library to provide a common resource for bovine genome research . We used leukocytes of a Japanese black bull (Bos taurus) as the DNA source, AB1380 for the yeast host, and pYAC4 for the vector . The library consists of 24,576 clones arranged in 256 96-well microtiter plates . An average insert size estimated from the analysis of 251 randomly selected clones was 480 kb . The rate of chimeric YACs evaluated by fluorescence in situ hybridization (FISH) analysis of 44 randomly selected clones was 36.4% . To estimate the number of genome equivalents, PCR-based screening was performed with 48 primer pairs and isolated 3.2 clones on average . In order to provide broad access for the scientific community, this library has been incorporated into the Reference Library system which provides high density filters for colony hybridization screening and a common database of the library.

Hokkaido Igaku Zasshi, 1998 May, 73(3), 275 - 86
{Analyses of p53 mutations in breast cancers with a combined use of yeast functional assay and immunohistochemical staining}; Takahashi M; p53 status was examined in a total of 52 primary breast cancers, using a combination of yeast-based assay for transcriptional activity of p53 (yeast functional assay, YA) and immunohistochemical staining (IHC) for p53 protein accumulation . Results by the two methods were compared, and their correlations to the clinicopathological characteristics were analyzed . Nineteen cases (37%) were shown to have p53 mutations by YA, while 11 (21%) were shown to be positive of p53 accumulation by IHC . The tumors were classified into 4 groups according to the results by the two methods: Group I (8/52), YA (+) & IHC (+): Group II (11/52), YA (+) & IHC (-): Group III (3/52), YA (-) & IHC (+) and Group IV (30/52), YA (-) & IHC (-) . DNA sequence analysis of all the YA (+) cases showed missense mutations in 7 out of 8 cases of Group I and nonsense or frameshift mutations in 8 out of 11 cases of Group II . The presence of p53 mutations significantly correlated with the absence of estrogen receptor in the tumors (Group I + II versus Group III + IV), whereas the number of stained cells positive of p53 did not . Microvascular invasion was significantly more frequent in Group I (75%) than in Group II (27%) (P < 0.05) . Survival of the patients assessed by Kaplan-Meier method was the best in Group III, followed by Group IV, II and I (P < 0.05) in that order . These results indicate that tumor p53 status determined by the yeast functional assay and immunohistochemistry can serve as an important prognosticator for patients with breast cancer.

Curr Opin Cell Biol, 1998 Aug, 10(4), 513 - 22
Protein traffic in the yeast endocytic and vacuolar protein sorting pathways; Wendland B et al.; Endocytosis is a fundamental membrane trafficking event that occurs in all eukaryotes . The yeast Saccharomyces cerevisiae has been particularly useful in efforts to uncover novel proteins that mediate endocytosis, and many of these factors share similarity with proteins from higher eukaryotes . In the past two years, progress has centered on three major areas: modifications/signaling pathways that initiate or regulate internalization, protein complexes that are implicated in the internalization process, and factors that are involved in regulation of traffic through late endosomal compartments . As the parallels between the mechanisms employed in yeast and higher eukaryotes are further explored, new insights into the complex process of endocytosis should emerge.

J Mol Biol, 1998 Sep 4, 281(5), 827 - 42
Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies; van Helden J et al.; We present here a simple and fast method allowing the isolation of DNA binding sites for transcription factors from families of coregulated genes, with results illustrated in Saccharomyces cerevisiae . Although conceptually simple, the algorithm proved efficient for extracting, from most of the yeast regulatory families analyzed, the upstream regulatory sequences which had been previously found by experimental analysis . Furthermore, putative new regulatory sites are predicted within upstream regions of several regulons . The method is based on the detection of over-represented oligonucleotides . A specificity of this approach is to define the statistical significance of a site based on tables of oligonucleotide frequencies observed in all non-coding sequences from the yeast genome . In contrast with heuristic methods, this oligonucleotide analysis is rigorous and exhaustive . Its range of detection is however limited to relatively simple patterns: short motifs with a highly conserved core . These features seem to be shared by a good number of regulatory sites in yeast . This, and similar methods, should be increasingly required to identify unknown regulatory elements within the numerous new coregulated families resulting from measurements of gene expression levels at the genomic scale . All tools described here are available on the web at the site yeast-tools

J Mol Biol, 1998 Sep 4, 281(5), 763 - 75
Structural and functional architecture of the yeast cell-cycle transcription factor swi6; Sedgwick SG et al.; The structural and functional organisation of Swi6, a transcriptional regulator of the budding yeast cell cycle has been analysed by a combination of biochemical, biophysical and genetic methods . Limited proteolysis indicates the presence of a approximately 15 kDa N-terminal domain which is dispensable for Swi6 activity in vivo and which is separated from the rest of the molecule by an extended linker of at least 43 residues . Within the central region, a 141 residue segment that is capable of transcriptional activation encompasses a structural domain of approximately 85 residues . In turn, this is tightly associated with an adjacent 28 kDa domain containing at least four ankyrin-repeat (ANK) motifs . A second protease sensitive region connects the ANK domain to the remaining 30 kDa C-terminal portion of Swi6 which contains a second transcriptional activator and sequences required for heteromerisation with Swi4 or Mbp1 . Transactivation by the activating regions of Swi6 is antagonised when either are combined with the central ankyrin repeat motifs . Hydrodynamic measurements indicate that an N-terminal 62 kDa fragment comprising the first three domains is monomeric in solution and exhibits an unusually high frictional coefficient consistent with the extended, multi-domain structure suggested by proteolytic analysis .

J Cell Sci, 1998 Sep, 111 ( Pt 18), 2799 - 807
Evidence for a novel MAPKKK-independent pathway controlling the stress activated Sty1/Spc1 MAP kinase in fission yeast; Shieh JC et al.; The fission yeast Sty1/Spc1 MAP kinase, like the mammalian JNK/SAPK and p38/CSBP1 kinases, is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, heat shock, UV light and the protein synthesis inhibitor anisomycin . Sty1 is activated by a single MAPKK, Wis1 . We demonstrate that the conserved MAPKKK phosphorylation sites Ser 469 and Thr 473 in the catalytic domain of Wis1 are normally essential for Sty1 activation . However, when mildly overexpressed, a mutant Wis1 kinase lacking these conserved phosphorylation sites is able to support stress inducible gene expression and activation of the Sty1 MAP kinase in response to an oxidative or osmotic stress or to a mild heat shock . We show that phosphorylation and activation of Sty1 under these conditions is not due to inactivation of the Pyp1 MAP kinase phosphatase . These results reveal a novel MAPKKK-independent pathway by which the Wis1 MAPKK can activate the Sty1 MAPK in response to stress in fission yeast.

Yeast, 1998 Jul, 14(10), 935 - 42
Drug-induced phenotypes provide a tool for the functional analysis of yeast genes; Launhardt H et al.; The post-genome sequencing era of Saccharomyces cerevisiae is defined by the analysis of newly discovered open reading frames of unknown function . In this report, we describe a genetic method for the rapid identification and characterisation of genes involved in a given phenotype . This approach is based on the ability of overexpressed genomic DNA fragments to cure an induced phenotype in yeast . To validate this concept, yeast cells carrying a yeast DNA library present on multicopy plasmid vectors were screened for resistance to the antifungal drug ketoconazole . Among 1.2 million colonies 13 clones tested positive, including those expressing the lanosterol C-14 demethylase, known to be a cellular target for azole drugs, and the cytochrome-c oxidase of mitochondria, regulating the respiratory chain electron transport . Several other resistant clones were identified, which code for yeast proteins of so far unknown function . These genes may represent potential candidates for antifungal drug effects . Together with the availability of the entire yeast genome sequence, the described genetic screening method is a powerful tool for the effective functional analysis of yeast genes.

Yeast, 1998 Jul, 14(10), 885 - 93
Differentiation of brewing yeast strains by pyrolysis mass spectrometry and Fourier transform infrared spectroscopy; Timmins EM et al.; Two rapid spectroscopic approaches for whole-organism fingerprinting--pyrolysis mass spectrometry (PyMS) and Fourier transform infrared spectroscopy (FT-IR)--were used to analyse 22 production brewery Saccharomyces cerevisiae strains . Multivariate discriminant analysis of the spectral data was then performed to observe relationships between the 22 isolates . Upon visual inspection of the cluster analyses, similar differentiation of the strains was observed for both approaches . Moreover, these phenetic classifications were found to be very similar to those previously obtained using genotypic studies of the same brewing yeasts . Both spectroscopic techniques are rapid (typically 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of the successful discrimination of both ale and lager yeasts . We believe that these whole-organism fingerprinting methods could find application in brewery quality control laboratories.

Mol Cell Biol, 1998 Sep, 18(9), 5000 - 9
CUS2, a yeast homolog of human Tat-SF1, rescues function of misfolded U2 through an unusual RNA recognition motif; Yan D et al.; A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins . CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a . Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly . Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2 . Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2 . Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.

Antonie Van Leeuwenhoek, 1998 Feb, 73(2), 163 - 8
Homothallic life cycle in the diploid red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma); Kucsera J et al.; Sexual activity was induced in the basidiomyceteous Phaffia rhodozyma (Xanthophyllomyces dendrorhous) by depletion of nitrogen from the culture medium . This activity involved both mating between two yeast cells and the formation of basidiospores . Mating is possibly started by a G1 phase arrest of the cell cycle, as in other yeasts . The life cycle exhibited homothallic features . Crosses between genetically marked strains, and pulse-field gel electrophoresis of the chromosomal DNA of cells derived from individual spores revealed evidence of karyogamy, meiosis and even recombination . The segregation ratio in tetrads pointed to diploid vegetative cells, which formed tetraploid zygotes and the immediate meiosis then gave rise to diploid progenies again . Apart from the type strain Phaffia rhodozyma CBS 5905, all the examined strains were able to sporulate.

Antonie Van Leeuwenhoek, 1998 Feb, 73(2), 143 - 6
Myxozyma neglecta sp . nov . (Candidaceae) a new yeast species from South Africa; Spaaij F et al.; Three strains of Myxozyma mucilagina including the type strain were reexamined . Based on differences in their carbon utilization pattern, mobility of isoenzymes, mol% G + C of their DNA and extent of DNA complementary the new species Myxozyma neglecta is proposed.

Genes Dev, 1998 Aug 15, 12(16), 2574 - 86
Telomere-mediated chromosome pairing during meiosis in budding yeast; Rockmill B et al.; Certain haploid strains of Saccharomyces cerevisiae can undergo meiosis, but meiotic prophase progression and subsequent nuclear division are delayed if these haploids carry an extra chromosome (i . e., are disomic) . Observations indicate that interactions between homologous chromosomes cause a delay in meiotic prophase, perhaps to allow time for interhomolog interactions to be completed . Analysis of meiotic mutants demonstrates that the relevant aspect of homolog recognition is independent of meiotic recombination and synaptonemal complex formation . A disome in which the extra chromosome is circular sporulates without a delay, indicating that telomeres are important for homolog recognition . Consistent with this hypothesis, fluorescent in situ hybridization demonstrates that a circular chromosome has a reduced capacity to pair with its homolog, and a telomere-associated meiotic protein (Ndj1) is required to delay sporulation in disomes . A circular dimer containing two copies of the same chromosome delays meiosis to the same extent as two linear homologs, implying that physical proximity bypasses the requirement for telomeres in homolog pairing . Analysis of a disome carrying two linear permuted chromosomes suggests that even nonhomologous chromosome ends can promote homolog pairing to a limited extent . We speculate that telomere-mediated chromosome movement and/or telomere clustering promote homolog pairing.

Genes Dev, 1998 Aug 15, 12(16), 2510 - 21
Activated transcription independent of the RNA polymerase II holoenzyme in budding yeast; McNeil JB et al.; We investigated whether the multisubunit holoenzyme complex of RNA polymerase II (Pol II) and mediator is universally required for transcription in budding yeast . DeltaCTD Pol II lacking the carboxy-terminal domain of the large subunit cannot assemble with mediator but can still transcribe the CUP1 gene . CUP1 transcripts made by DeltaCTD Pol II initiated correctly and some extended past the normal poly(A) site yielding a novel dicistronic mRNA . Most CUP1 transcripts made by DeltaCTD Pol II were degraded but could be stabilized by deletion of the XRN1 gene . Unlike other genes, transcription of CUP1 and HSP82 also persisted after inactivation of the CTD kinase Kin28 or the mediator subunit Srb4 . The upstream-activating sequence (UAS) of the CUP1 promoter was sufficient to drive Cu2+ inducible transcription without Srb4 and heat shock inducible transcription without the CTD . We conclude that the Pol II holoenzyme is not essential for all UAS-dependent activated transcription in yeast.

Biotechniques, 1998 Aug, 25(2), 294 - 6
Highly efficient oligonucleotide transfer into intact yeast cells using square-wave pulse electroporation; Barre FX et al.; Here, we present a rapid and reproducible procedure based on square-wave pulse electroporation that allows efficient penetration of synthetic oligonucleotides into intact yeast cells . This procedure was successfully used to modify the yeast genome with small amounts of oligonucleotide.

Biochim Biophys Acta, 1998 Aug 14, 1404(1-2), 211 - 30
Multiple sorting pathways between the late Golgi and the vacuole in yeast; Conibear E et al.; Newly synthesized proteins that reach the last compartment of the Golgi complex can be sorted into pathways leading either to the cell surface or to the vacuole . It now appears that there are at least two routes from the Golgi to the vacuole: the 'CPY pathway', which involves transit through an endosomal/prevacuolar compartment (PVC), and a recently discovered 'ALP pathway', which bypasses the PVC, but may involve other as yet unidentified intermediate compartments . No cytosolic signal has been identified that directs the entry of membrane proteins into the CPY pathway . In contrast, the transport of ALP through the ALP pathway is saturable and signal mediated . Much recent work has focused on the identification of proteins that regulate trafficking to the vacuole . A number of genes have been identified that are specific for either the CPY or ALP sorting pathways, while other genes affect both types of transport and may therefore act at or after a point of convergence . Progress has also been made in further elucidating the members of the SNARE complexes that act in Golgi-to-PVC transport as well as those that mediate fusion with the vacuole.

Biochim Biophys Acta, 1998 Aug 14, 1404(1-2), 33 - 51
COPI in ER/Golgi and intra-Golgi transport: do yeast COPI mutants point the way?
Gaynor EC, Graham TR, Emr SD.
Coat complexes facilitate the formation of transport vesicles which are essential for proper trafficking of protein and lipids through the secretory pathway . Since its initial identification in the mid-1980s, the COPI coat complex has been credited with mediating multiple distinct transport events and intracellular processes in the exocytic pathway . Not surprisingly, the diversity of these functions has led to significant debate concerning the primary function of COPI . Specifically, within the ER/Golgi and intra-Golgi systems, does COPI mediate anterograde protein transport, retrograde protein transport, or both? This review will focus on the in vivo roles of COPI, primarily examining data from studies of yeast COPI mutants but also including evidence from mammalian systems as appropriate . Some of the current controversies surrounding whether COPI acts directly or indirectly in anterograde and retrograde transport will also be addressed . Because recruitment of COPI to membranes requires the small GTP-binding protein ARF, we will also discuss ARF and proteins that regulate ARF function, and how these proteins might modulate both COPI-driven events and overall membrane composition . Finally, we will point out some of the links still missing from our understanding of COPI-driven events and discuss possible future directions for studies of COPI function.

J Parasitol, 1998 Aug, 84(4), 870 - 2
The effects of dietary yeast on the cellular immune response of Drosophila melanogaster against the larval parasitoid, Leptopilina boulardi; Vass E et al.; The role of dietary yeast in Drosophila melanogaster cellular immunity was investigated . Host larvae deprived of yeast immediately after parasitization by the cynipid wasp Leptopilina boulardi encapsulated a significantly lower percentage of the parasitoid's eggs than hosts transferred to a medium with yeast . When the transfers of hosts were made 24 hr after exposure to the parasite, diet had no effect on the immune response that had commenced prior to the transfers . This study demonstrates for the first time the effect of a specific dietary component on the immune responsiveness of Drosophila against a larval parasitoid.

Arch Dis Child Fetal Neonatal Ed, 1998 May, 78(3), F225 - 6
Enteral yeast-selenium supplementation in preterm infants; Bogye G et al.; AIM: To study the bioavailability of selenium enriched yeast in preterm infants living in a low selenium area (Hungary) . METHODS: Thirty six preterm infants were randomly assigned to two groups at birth with respect to selenium supplementation . In the supplemented group (n = 18) infants received 4.8 mg of selenium enriched yeast containing 5 micrograms selenium daily . RESULTS: In the supplemented group the serum selenium concentration increased from 36.1 (+/- 12.8) micrograms/l to 43.5 (7.9) micrograms/l and in the non-supplemented group it decreased from 34.4 (20.4) micrograms/l to 26.1 (16.6) micrograms/l from birth in two weeks . No complications or side effects as a result of supplementation were observed . CONCLUSIONS: Selenium enriched yeast is a safe and an effective form of short term enteral selenium supplementation for preterm infants.

J Biol Chem, 1998 Aug 28, 273(35), 22595 - 605
Yeast transcript elongation factor (TFIIS), structure and function . II: RNA polymerase binding, transcript cleavage, and read-through; Awrey DE et al.; The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region . We have probed the function of this fragment of TFIIS using structure-guided mutagenesis . The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix . TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action . The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation . Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through . Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities . Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.

J Biol Chem, 1998 Aug 28, 273(35), 22589 - 94
Yeast transcript elongation factor (TFIIS), structure and function . I: NMR structural analysis of the minimal transcriptionally active region; Olmsted VK et al.; TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription . We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III) . The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation . Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III . We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct . We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS . The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure . NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.

J Biol Chem, 1998 Aug 28, 273(35), 22563 - 9
Interaction of BTG1 and p53-regulated BTG2 gene products with mCaf1, the murine homolog of a component of the yeast CCR4 transcriptional regulatory complex; Rouault JP et al.; Both BTG1 and BTG2 are involved in cell-growth control . BTG2 expression is regulated by p53, and its inactivation in embryonic stem cells leads to the disruption of DNA damage-induced G2/M cell-cycle arrest . In order to investigate the mechanism underlying Btg-mediated functions, we looked for possible functional partners of Btg1 and Btg2 . Using yeast two-hybrid screening, protein-binding assays, and transient transfection assays in HeLa cells, we demonstrated the physical in vitro and in vivo interaction of both Btg1 and Btg2 with the mouse protein mCaf1 (i.e . mouse CCR4-associated factor 1) . mCaf1 was identified through its interaction with the CCR4 protein, a component of a general transcription multisubunit complex, which, in yeast, regulates the expression of different genes involved in cell-cycle regulation and progression . These data suggest that Btg proteins, through their association with mCaf1, may participate, either directly or indirectly, in the transcriptional regulation of the genes involved in the control of the cell cycle . Finally, we found that box B, one of two conserved domains which define the Btg family, plays a functional role, namely that it is essential to the Btg-mCaf1 interaction.

J Biol Chem, 1998 Aug 28, 273(35), 22415 - 9
Site-directed mutagenesis of the yeast multicopper oxidase Fet3p; Askwith CC et al.; High affinity iron transport in yeast is mediated by two proteins, Fet3p and Ftr1p . The multicopper oxidase Fet3p is thought to convert extracellular ferrous iron to ferric iron, which then crosses the plasma membrane through the permease Ftr1p . Fet3p is capable of oxidizing other substrates, such as p-phenylenediamine, and there is still a question of whether it is the ferroxidase activity that is essential for iron transport . Fet3p is also required for Ftr1p localization to the cell surface, making it difficult to prove a direct role for Fet3p oxidase in high affinity iron transport . In an attempt to generate Fet3p specifically lacking ferroxidase activity, we used site-directed mutagenesis to alter residues within Fet3p that had been suggested to impart iron oxidase activity . These substitutions resulted in either a loss or retention of both p-phenylenediamine and ferroxidase activities, indicating that the ability of Fet3p to act as a ferroxidase involves other amino acids . Inactive Fet3p, however, did mediate Ftr1p localization to the cell surface but did not mediate high affinity iron transport . These observations indicate that the ferroxidase activity of Fet3p is intrinsically required for high affinity iron transport.

J Biol Chem, 1998 Aug 28, 273(35), 22181 - 7
Defects in the yeast high affinity iron transport system result in increased metal sensitivity because of the increased expression of transporters with a broad transition metal specificity; Li L et al.; Yeast with defects in vacuolar pH show increased sensitivity to high concentrations of transition metals . This sensitivity has been presumed to result from defective metal storage . We demonstrate that mutations that result in a defective high affinity iron transport system, such as a deletion in the surface ferroxidase FET3, also result in increased metal sensitivity independent of vacuolar function . Multiple copies of transition metal transporter resistance genes, such as COT1 or ZRC1, do not reduce the metal sensitivity of fet3 mutations . Increased metal sensitivity is because of an increased cellular accumulation of transition metals resulting from the increased activity of low affinity iron transporters, such as FET4, that mediates the transport of other transition metals . In cells lacking a high affinity iron transport system, the increased transition metal uptake can be prevented by increased extracellular iron . These results suggest that vacuolar function may not be required for transition metal sequestration.

Biochem Biophys Res Commun, 1998 Aug 19, 249(2), 531 - 6
Mutagenesis of human Mel1a melatonin receptor expressed in yeast reveals domains important for receptor function; Kokkola T et al.; A yeast functional colorimetric assay was employed to test the effects of site-directed point mutations on the function of the human Mel1a melatonin receptor . Seven mutants were created in transmembrane domains III, V, and VII of the receptor to test the rhodopsin-based model of melatonin recognition . Two mutants in transmembrane domains III and VI were created to investigate the mechanisms of G protein activation in the melatonin receptor . Mutations in transmembrane domain V either potentiated agonist efficiencies (H195A) or totally abolished all responses to tested compounds (V192T+H195A) . Mutation N124A in the conserved NRY motif in the end of transmembrane domain III seriously impaired receptor activation . Several mutants were found to have decreased ability to activate functional responses, reflecting the importance of these residues for receptor function . These data also suggest that activation of the receptor involves interaction of the 5-methoxy group of melatonin with the conserved histidine H195 in transmembrane domain V .

Methods Enzymol, 1998, 292, 193 - 212
Functional assays for analysis of yeast ste6 mutants; Nijbroek GL et al.; As a member of the ABC superfamily, STE6 is unique in that it has a well-characterized substrate, a-factor, and can be easily manipulated in the yeast system . Functional assays have been extensively used, and methods to examine trafficking and stability of STE6 are well established . In addition, STE6 chimeras and ste6 deletion strains are useful for the analysis of many nonyeast ABC proteins . Continuing studies of STE6 are expected to aid in the identification of novel cellular components involved in the trafficking and functioning of not only STE6, but of other members of the ABC superfamily as well.

Mol Cell Biol, 1998 Sep, 18(9), 5485 - 91
Functional and physical interaction between Rad24 and Rfc5 in the yeast checkpoint pathways; Shimomura T et al.; The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage . Here we describe the isolation of RAD24, known to play a role in the DNA damage checkpoint, as a dosage-dependent suppressor of rfc5-1 . RAD24 overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation . Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage . The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5 . Although the rad24Delta mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect in rfc5-1 mutants . Furthermore, overexpression of RAD24 suppresses the rfc5-1 defect in the replication block checkpoint . Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.

Mol Cell Biol, 1998 Sep, 18(9), 5308 - 19
Vam7p, a SNAP-25-like molecule, and Vam3p, a syntaxin homolog, function together in yeast vacuolar protein trafficking; Sato TK et al.; A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y . Wada and Y . Anraku, J . Biol . Chem . 267:18671-18675, 1992; T . Weimbs, S . H . Low, S . J . Chapin, K . E . Mostov, P . Bucher, and K . Hofmann, Proc . Natl . Acad . Sci . USA 94:3046-3051, 1997) . Analysis of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7(tsf)) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport . vam7(tsf) mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways . Examination of vam7(tsf) cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates . A fraction of Vam7p was localized to vacuolar membranes . Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3 . Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p . In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40(phox) (PX) domain . Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3(tsf) mutation, suggesting that the PX domain is required for Vam7p function . This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.

Mol Cell Biol, 1998 Sep, 18(9), 5189 - 98
Genetic, physical, and functional interactions between the triphosphatase and guanylyltransferase components of the yeast mRNA capping apparatus; Ho CK et al.; We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme) . CES5 is identical to CET1, which encodes the RNA triphosphatase component of the yeast capping apparatus . Purified recombinant Cet1 catalyzes hydrolysis of the gamma phosphate of triphosphate-terminated RNA at a rate of 1 s-1 . Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer . The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1 . This stimulation is the result of increased affinity for the GTP substrate . A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo . The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo . These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary . The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo . A mutation of the triphosphatase active-site cysteine of Mce1 is lethal . Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth . This work highlights the potential for regulating mRNA cap formation through protein-protein interactions.

Mol Cell Biol, 1998 Sep, 18(9), 5062 - 72
Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant; Boeck R et al.; mRNA in the yeast Saccharomyces cerevisiae is primarily degraded through a pathway that is stimulated by removal of the mRNA cap structure . Here we report that a mutation in the SPB8 (YJL124c) gene, initially identified as a suppressor mutation of a poly(A)-binding protein (PAB1) gene deletion, stabilizes the mRNA cap structure . Specifically, we find that the spb8-2 mutation results in the accumulation of capped, poly(A)-deficient mRNAs . The presence of this mutation also allows for the detection of mRNA species trimmed from the 3' end . These data show that this Sm-like protein family member is involved in the process of mRNA decapping, and they provide an example of 3'-5' mRNA degradation intermediates in yeast.

Mol Cell Biol, 1998 Sep, 18(9), 4971 - 6
Yeast coactivator MBF1 mediates GCN4-dependent transcriptional activation; Takemaru K et al.; Transcriptional coactivators play a crucial role in gene expression by communicating between regulatory factors and the basal transcription machinery . The coactivator multiprotein bridging factor 1 (MBF1) was originally identified as a bridging molecule that connects the Drosophila nuclear receptor FTZ-F1 and TATA-binding protein (TBP) . The MBF1 sequence is highly conserved across species from Saccharomyces cerevisiae to human . Here we provide evidence acquired in vitro and in vivo that yeast MBF1 mediates GCN4-dependent transcriptional activation by bridging the DNA-binding region of GCN4 and TBP . These findings indicate that the coactivator MBF1 functions by recruiting TBP to promoters where DNA-binding regulators are bound.

J Mol Biol, 1998 Aug 28, 281(4), 631 - 49
Semi-conservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template; Braguglia D et al.; We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro . This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated {alpha-32P}dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with the heavy derivative BrdUTP, which results in a shift in density corresponding to complete second strand synthesis . We demonstrate dependence on DNA pol delta and the pol alpha/primase complex, and are able to detect putative Okazaki fragments under ATP-limiting conditions . In contrast to the semi-conservative replication of supercoiled plasmid, linear or open-circular templates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients . Consistent with a true replication reaction we find that semi-conservative replication of plasmid DNA is stimulated in S-phase relative to G1-phase nuclear extracts, and is independent of the recombination-promoting factor Rad52p . Using this novel system we demonstrate that semi-conservative replication, but not polymerase activity per se, requires the activity of the DNA helicase encoded by DNA2 .

Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 10009 - 14
A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing mRNAs in mammalian cells; Sun X et al.; All eukaryotic cells analyzed have developed mechanisms to eliminate the production of mRNAs that prematurely terminate translation . The mechanisms are thought to exist to protect cells from the deleterious effects of in-frame nonsense codons that are generated by routine inefficiencies and inaccuracies in RNA metabolism such as pre-mRNA splicing . Depending on the particular mRNA and how it is produced, nonsense codons can mediate a reduction in mRNA abundance either (i) before its release from an association with nuclei into the cytoplasm, presumably but not certainly while the mRNA is being exported to the cytoplasm and translated by cytoplasmic ribosomes, or (ii) in the cytoplasm . Here, we provide evidence for a factor that functions to eliminate the production of nonsense-containing RNAs in mammalian cells . The factor, variously referred to as Rent1 (regulator of nonsense transcripts) or HUPF1 (human Upf1 protein), was identified by isolating cDNA for a human homologue to Saccharomyces cerevisiae Upf1p, which is a group I RNA helicase that functions in the nonsense-mediated decay of mRNA in yeast . Using monkey COS cells and human HeLa cells, we demonstrate that expression of human Upf1 protein harboring an arginine-to-cysteine mutation at residue 844 within the RNA helicase domain acts in a dominant-negative fashion to abrogate the decay of nonsense-containing mRNA that takes place (i) in association with nuclei or (ii) in the cytoplasm . These findings provide evidence that nonsense-mediated mRNA decay is related mechanistically in yeast and in mammalian cells, regardless of the cellular site of decay.

EMBO J, 1998 Aug 17, 17(16), 4873 - 80
The yeast Ty3 retrotransposon contains a 5'-3' bipartite primer-binding site and encodes nucleocapsid protein NCp9 functionally homologous to HIV-1 NCp7; Gabus C et al.; Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication . During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome . Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site . Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet . Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems . Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs.

EMBO J, 1998 Aug 17, 17(16), 4859 - 72
A novel yeast gene, THO2, is involved in RNA pol II transcription and provides new evidence for transcriptional elongation-associated recombination; Piruat JI et al.; We have identified two novel yeast genes, THO1 and THO2, that partially suppress the transcription defects of hpr1Delta mutants by overexpression . We show by in vivo transcriptional and recombinational analysis of tho2Delta cells that THO2 plays a role in RNA polymerase II (RNA pol II)-dependent transcription and is required for the stability of DNA repeats, as previously shown for HPR1 . The tho2Delta mutation reduces the transcriptional efficiency of yeast DNA sequences down to 25% of the wild-type levels and abolishes transcription of the lacZ sequence . In addition, tho2Delta causes a strong increase in the frequency of recombination between direct repeats (>2000-fold above wild-type levels) . Some DNA repeats cannot even be maintained in the cell . This hyper-recombination phenotype is dependent on transcription and is not observed in DNA repeats that are not transcribed . The higher the impairment of transcription caused by tho2Delta, the higher the frequency of recombination of a particular DNA region . The tho2Delta mutation also increases the frequency of plasmid loss . Our work not only identifies a novel yeast gene, THO2, with similar function to HPR1, but also provides new evidence for transcriptional blocks as a source of recombination . We propose that there is a set of proteins including Hpr1p and Tho2p, in the absence of which RNA pol II transcription is stalled or blocked, causing genetic instability.

EMBO J, 1998 Aug 17, 17(16), 4798 - 808
Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20; Ptushkina M et al.; Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far . This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding . Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth . The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity . Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity . The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E . Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein . The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.

EMBO J, 1998 Aug 17, 17(16), 4780 - 9
Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2; Gerber A et al.; We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine . The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested . Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37 . Transformation of the mutant cells with the TAD1 gene restored enzyme activity . Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA . These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification.

Curr Biol, 1998 Jul 30-Aug 13, 8(16), 927 - 30
A role for the yeast actin cytoskeleton in pheromone receptor clustering and signalling; Ayscough KR et al.; The development of cell polarity in response to external stimuli is a feature of most eukaryotic cell types . Haploid cells of the budding yeast Saccharomyces cerevisiae secrete peptide pheromones to induce conjugation with cells of the opposite mating type . Pheromone binding triggers transcription of mating-specific genes, cell cycle arrest in the G1 phase and the formation of a mating projection oriented toward the source of pheromone {1-2} . Based on a multitude of studies in diverse eukaryotic cells, it has been hypothesized that hierarchies of proteins function to govern the generation of cell polarity {3-4} . Numerous proteins have been identified in yeast that accumulate both at a position on the cell cortex that will develop into a mating projection in response to pheromone binding and at the site of bud formation in response to an intrinsic cue during mitotic growth . When the actin cytoskeleton is disrupted before bud formation by the addition of latrunculin-A (LAT-A), several proteins involved in budding, including the GTPase Cdc42p, are still able to achieve their appropriate polarized localization {5} . In contrast, we show here that following pheromone addition, an intact actin cytoskeleton is required for localization of several proteins to a discrete position on the cell cortex . We also demonstrate a role for actin in pheromone-induced receptor clustering and signalling . We propose that actin-mediated pheromone receptor clustering might consolidate signalling from Cdc42p to one region of the cell cortex so that small differences in receptor occupancy across the cell surface can be amplified into dramatic cellular polarity.

Nucleic Acids Res, 1998 Sep 1, 26(17), 3977 - 85
Mutations in the yeast Myb-like protein Bas1p resulting in discrimination between promoters in vivo but notin vitro; Pinson B et al.; Bas1p is a yeast transcription factor that activates expression of purine and histidine biosynthesis genes in response to extracellular purine limitation . The N-terminal part of Bas1p contains an Myb-like DNA binding domain composed of three tryptophan-rich imperfect repeats . We show that mutating the conserved tryptophan residues in the DNA binding domain of Bas1p severely impairs in vivo activation of target genes and in vitro DNA binding of Bas1p . We also found that two mutations (H34L and W42A) in the first repeat make Bas1p discriminate between promoters in vivo . These two BAS1 mutants are able to activate expression of an HIS4-lacZ fusion but not that of ADE1-lacZ or ADE17-lacZ fusions . Surprisingly, these mutant proteins bind equally well to the three promoters in vitro , suggesting that the mutations affect the interaction of Bas1p with some promoter-specific factor(s) in vivo . By mutating a potential nucleotide binding site in the DNA binding domain of Bas1p, we also show that this motif does not play a major role in purine regulation of Bas1p activity . Finally, using a green fluorescence protein (GFP)-Bas1p fusion, we establish the strict nuclear localization of Bas1p and show that it is not affected by extracellular adenine.

Nucleic Acids Res, 1998 Sep 1, 26(17), 3971 - 6
HRAD1 and MRAD1 encode mammalian homologues of the fission yeast rad1(+) cell cycle checkpoint control gene; Udell CM et al.; Eukaryotic cells arrest at the G2checkpoint in the presence of DNA damage or incompletely replicated DNA . This cell cycle checkpoint prevents the development and propagation of genomic instability . In the fission yeast, this process requires the action of a number of genes, including rad1(+) . We report here the identification of human and mouse cDNAs that exhibit extensive sequence homology to rad1(+) . The human gene, called HRAD1 , encodes a 282 amino acid protein that is 27% identical and 53% similar to yeast Rad1p . The human homologue maintains its sequence similarity over the full length of the protein, including the three proposed 3'-->5' exonuclease domains, and the leucine rich repeat region . The mouse gene, called MRAD1 , encodes a 280 amino acid protein that is 90% identical and 96% similar to HRAD1 at the amino acid level . Expression of HRAD1 in yeast rad1 mutants partially restores radiation resistance and G2checkpoint proficiency to these mutants . Evolutionaryconservation of structure between HRAD1 , MRAD1 , rad1(+), Saccharomyces cerevisiae RAD17 and the Ustilago maydis REC1 checkpoint genes suggests that the function of the encoded proteins is conserved as well . The ability of HRAD1 to partially complement yeast rad1 mutants suggests that this gene is required for G2checkpoint control in human cells.

Nucleic Acids Res, 1998 Sep 1, 26(17), 3908 - 14
The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) define a structurally and functionally conserved Ubc-like protein family; Xiao W et al.; Eukaryotic genes encoding ubiquitin-congugating enzyme (Ubc)-like proteins have been isolated from both human and yeast cells . The CROC-1 gene was isolated by its ability to transactivate c- fos expression in cell culture through a tandem repeat enhancer sequence . The yeast MMS2 gene was cloned by its ability to complement the methyl methanesulfonate sensitivity of the mms2-1 mutant and was later shown to be involved in DNA post-replication repair . We report here the identification of a human MMS2 ( hMMS2 ) cDNA encoding a novel human Ubc-like protein . hMMS2 and CROC-1 share >90% amino acid sequence identity, but their DNA probes hybridize to distinct transcripts . hMMS2 and CROC-1 also share approximately 50% identity and 75% similarity with the entire length of yeast Mms2 . Unlike CROC-1 , whose transcript appears to be elevated in all tumor cell lines examined, the hMMS2 transcript is only elevated in some tumor cell lines . Collectively, these results indicate that eukaryotic cells may contain a highly conserved family of Ubc-like proteins that play roles in diverse cellular processes, ranging from DNA repair to signal transduction and cell differentiation . The hMMS2 and CROC-1 genes are able to functionally complement the yeast mms2 defects with regard to sensitivity to DNA damaging agents and spontaneous mutagenesis . Conversely, both MMS2 and hMMS2 were able to transactivate a c- fos - CAT reporter gene in Rat-1 cells in a transient co-transfection assay . We propose that either these proteins function in a common cellular process, such as DNA repair, or they exert their diverse biological roles through a similar biochemical interaction relative to ubiquitination.

J Biol Chem, 1998 Aug 21, 273(34), 22083 - 90
Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast; Brown GW et al.; Members of the Cdc7 family of protein kinases are essential for the initiation of DNA replication in all eukaryotes, but their precise biochemical function is unclear . We have purified the fission yeast Cdc7 homologue Hsk1 approximately 30,000-fold, to near homogeneity . Purified Hsk1 has protein kinase activity on several substrates and is capable of autophosphorylation . Point mutations in highly conserved regions of Hsk1 inactivate the kinase in vitro and in vivo . Overproduction of two of the mutant hsk1 alleles blocks initiation of DNA replication and deranges the mitotic checkpoint, a phenotype consistent with a role for Hsk1 in the early stages of initiation . The purified Hsk1 kinase can be separated into two active forms, a Hsk1 monomer and a heterodimer consisting of Hsk1 complexed with a co-purifying polypeptide, Dfp1 . Association with Dfp1 stimulates phosphorylation of exogenous substrates but has little effect on autokinase activity . We have identified Dfp1 as the fission yeast homologue of budding yeast Dbf4 . Purified Hsk1 phosphorylates the Cdc19 (Mcm2) subunit of the six-member minichromosome maintenance protein complex purified from fission yeast . Since minichromosome maintenance proteins have been implicated in the initiation of DNA replication, the essential function of Hsk1 at the G1/S transition may be mediated by phosphorylation of Cdc19 . Furthermore, the phosphorylation of critical substrates by Hsk1 kinase is likely regulated by association with a Dbf4-like co-factor.

J Biol Chem, 1998 Aug 21, 273(34), 21972 - 9
Characterization of the CP complex, an abundant dimer of Cdc68 and Pob3 proteins that regulates yeast transcriptional activation and chromatin repression; Brewster NK et al.; The Cdc68 protein of the yeast Saccharomyces cerevisiae is an essential nuclear protein that has been shown to be necessary for the trans-activation of many genes as well as for the maintenance of chromatin-mediated repression in the absence of trans-activation . These activities implicate the Cdc68 protein in the regulation of chromatin structure and/or function . Here we report that Cdc68 is found in association with another essential nuclear protein, Pob3, in what we term the CP complex . This dimer of Cdc68 with Pob3 is stable to partial purification, so that the functions of gene activation and repression that are assigned to Cdc68 are likely to be properties of the CP complex . The CP complex is highly abundant, suggesting that it may be widespread throughout chromatin.

J Biol Chem, 1998 Aug 21, 273(34), 21744 - 51
Phosphorylation region of the yeast plasma-membrane H+-ATPase . Role in protein folding and biogenesis; DeWitt ND et al.; Mutations at the phosphorylation site (Asp-378) of the yeast plasma-membrane H+-ATPase have been shown previously to cause misfolding of the ATPase, preventing normal movement along the secretory pathway; Asp-378 mutations also block the biogenesis of co-expressed wild-type ATPase and lead to a dominant lethal phenotype . To ask whether these defects are specific for Asp-378 or whether the phosphorylation region as a whole is involved, alanine-scanning mutagenesis has been carried out to examine the role of 11 conserved residues flanking Asp-378 . In the sec6-4 expression system (Nakamoto, R . K., Rao, R., and Slayman, C . W . (1991) J . Biol . Chem . 266, 7940-7949), the mutant ATPases displayed varying abilities to reach the secretory vesicles that deliver plasma-membrane proteins to the cell surface . Indirect immunofluorescence of intact cells also gave evidence for a spectrum of behavior, ranging from mutant ATPases completely arrested (D378A, K379A, T380A, and T384A) or partially arrested in the endoplasmic reticulum to those that reached the plasma membrane in normal amounts (C376A, S377A, and G381A) . Although the extent of ER retention varied among the mutants, the endoplasmic reticulum appeared to be the only secretory compartment in which the mutant ATPases accumulated . All of the mutant proteins that localized either partially or fully to the ER were also malfolded based on their abnormal sensitivity to trypsin . Among them, the severely affected mutants had a dominant lethal phenotype, and even the intermediate mutants caused a visible slowing of growth when co-expressed with wild-type ATPase . The effects on growth could be traced to the trapping of the wild-type enzyme with the mutant enzyme in the ER, as visualized by double label immunofluorescence . Taken together, the results indicate that the residues surrounding Asp-378 are critically important for ATPase maturation and transport to the cell surface.

J Biol Chem, 1998 Aug 21, 273(34), 21526 - 30
Peptides glycosylated in the endoplasmic reticulum of yeast are subsequently deglycosylated by a soluble peptide: N-glycanase activity; Suzuki T et al.; Several lines of evidence suggest that soluble peptide:N-glycanase (PNGase) is involved in the quality control system for newly synthesized glycoproteins in mammalian cells . Here we report the occurrence of a soluble PNGase activity in Saccharomyces cerevisiae . The enzyme, which was recovered in the cytosolic fraction, has a neutral pH optimum, and dithiothreitol is required for activity . All of these properties were similar to those of earlier described for mammalian PNGases . Interestingly, the yeast enzyme activity was found to be present almost exclusively in cells in stationary phase; little activity was detected in logarithmic growth phase cells . Upon incubation of a glycosylatable peptide R-Asn-X-Thr-R' with permeabilized yeast spheroplasts, we detected formation of both glycosylated peptide and the peptide product expected from PNGase-mediated deglycosylation of this glycopeptide, namely, R-Asp-X-Thr-R' . Recent findings that yeast have an active system for the retrograde transport of unfolded (glyco)proteins and glycopeptides out of the endoplasmic reticulum (ER) into the cytosol raise the possibility that this PNGase may participate in an early step in degradation of these molecules following their export from the ER.

J Biol Chem, 1998 Aug 21, 273(34), 21489 - 96
A yeast homologue of the human phosphotyrosyl phosphatase activator PTPA is implicated in protection against oxidative DNA damage induced by the model carcinogen 4-nitroquinoline 1-oxide; Ramotar D et al.; The model carcinogen 4-nitroquinoline 1-oxide (4-NQO) has historically been characterized as "UV-mimetic" with respect to its genotoxic properties . However, recent evidence indicates that 4-NQO, unlike 254-nm UV light, may exert significant cytotoxic and/or mutagenic potential via the generation of reactive oxygen species . To elucidate the response of eukaryotic cells to 4-NQO-induced oxidative stress, we isolated Saccharomyces cerevisiae mutants exhibiting hypersensitivity to the cytotoxic effects of this mutagen . One such mutant, EBY1, was cross-sensitive to the oxidative agents UVA and diamide while retaining parental sensitivities to 254-nm UV light, methyl methanesulfonate, and ionizing radiation . A complementing gene (designated yPTPA1), restoring full UVA and 4-NQO resistance to EBY1 and encoding a protein that shares 40% identity with the human phosphotyrosyl phosphatase activator hPTPA, has been isolated . Targeted deletion of yPTPA1 in wild type yeast engendered the identical pattern of mutagen hypersensitivity as that manifested by EBY1, in addition to a spontaneous mutator phenotype that was markedly enhanced upon exposure to either UVA or 4-NQO but not to 254-nm UV or methyl methanesulfonate . Moreover, the yptpa1 deletion mutant exhibited a marked deficiency in the recovery of high molecular weight DNA following 4-NQO exposure, revealing a defect at the level of DNA repair . These data (i) strongly support a role for active oxygen intermediates in determining the genotoxic outcome of 4-NQO exposure and (ii) suggest a novel mechanism in yeast involving yPtpa1p-mediated activation of a phosphatase that participates in the repair of oxidative DNA damage, implying that hPTPA may exert a similar function in humans.

Biol Chem, 1998 Jul, 379(7), 831 - 40
Yeast elongation factor 3: structure and function; Chakraburtty K et al.; Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus . EF-3 is a single polypeptide protein with a molecular weight of 116,000 required by yeast ribosomes for in vitro translation and for in vivo growth . The YEF3 gene, located on chromosome xii, is essential for the survival of yeast . The deduced amino acid sequence of EF-3 has revealed the presence of duplicated ATP-binding cassettes similar to those present in the membrane associated transporters . The carboxy-terminus of EF-3 contains blocks of lysine boxes essential for its functional interaction with yeast ribosomes . EF-3 stimulates binding of aminoacyl-tRNA to the ribosomal A-site by facilitating release of deacylated tRNA from the exit site (E-site) . Chasing experiments revealed that EF-3 enhances the rate of tRNA dissociation from the E-site by a factor of two without affecting the affinity of the site for tRNA . EF-3 function is dependent on ATP hydrolysis . The existence of functional homologs of EF-3 in higher eukaryotes is still an open question . Further investigations are needed to settle this issue.

Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 621 - 7
The nonactive site cysteine residues of yeast protein disulfide isomerase are not required for cell viability; Luz JM et al.; Protein disulfide isomerase (PDI), the product of the essential PDI1 gene of Saccharomyces cerevisiae catalyzes oxidization of thiols, reduction of disulfide bonds, and isomerization of disulfides . It can also act as a chaperone to facilitate folding of denatured proteins . The protein has 6 cysteine (Cys) residues . Four of these Cys are part of the 2 thioredoxin-like catalytic sites (-CGHC-), one of which is located near the N- and the other near the C-terminus . In addition, it has 2 non-active site Cys near the N-terminus . The function of these non-active site Cys of yeast PDI is poorly understood . Whereas in yeast PDI, these Cys residues are in the vicinity of the N-terminal-most active site, in mammalian PDI their position is closer to the C-terminal-most active site . We have examined their role and that of the active site cysteines by constructing an extensive set of mutants in which the Cys were systematically replaced by Ser . As reported earlier, the N-terminal Cys of the two active sites sequences of yeast PDI were found to be required for cell viability, but mutation of the C-terminal Cys to Ser in the two active sites was not lethal . We found that replacement of the two non-active site Cys with Ser did not affect cell viability, but in the case of the double mutant in which both Cys were replaced by Ser the processing and secretion of CPY was impaired.

Environ Health Perspect, 1998 Aug, 106 Suppl 4, 1027 - 32
Analysis of repair and mutagenesis of chromium-induced DNA damage in yeast, mammalian cells, and transgenic mice; Cheng L et al.; Chromium (Cr) is a widespread environmental contaminant and a known human carcinogen . We have used shuttle vector systems in yeast, mammalian cells, and transgenic mice to characterize the mutational specificity and premutational DNA damage induced by Cr(VI) and its reduction intermediates in order to elucidate the mechanism by which Cr induces mutations . In the yeast system, treatment of vector-containing cells with Cr(VI) results in a dose-dependent increase in mutations in the SUP4-o target gene of the vector; mutagenesis is enhanced in an apn-1 yeast mutant, deficient in the capacity to repair oxidative-type DNA damage . In vector-containing mammalian cells, treatment with Cr(VI) also results in a dose-dependent increase in mutations in the vector target gene supF . The Cr-induced mutations in supF occurred mostly at G:C base pairs and were widely distributed across the gene, a pattern similar to those observed with ionizing radiation or hydrogen peroxide . These results support the hypothesis that Cr(VI)-induced oxidative-type DNA damage is responsible for Cr mutagenesis in the cell . Recently these studies were extended into the Big Blue transgenic mouse system in which Cr-induced mutagenesis was observed in the lung, the target organ for Cr carcinogenesis in humans . Analysis of the spectrum of these mutations will test whether Cr mutagenesis occurs by similar mechanisms in the intact animal as in cell culture systems and yeast.

Biofizika, 1998 May-Jun, 43(3), 447 - 51
{Study of fluorescence from isolated yeast plasma membranes in visible range of the spectrum}; Strakhovskaia MG et al.; The fluorescence with maximum at 683 nm from isolated yeast plasma membranes has been detected . The fluorescence was due to a membrane-bound compound absorbing in the visible range of the spectrum . The fluorescence excitation spectrum of this compound has a structure typical for porphyrins . At the same time, in several fluorescence properties the porphyrin localized in the plasma membrane is different from other intracellular porphyrins (protoporphyrin, coproporphyrin).

Plant Physiol, 1998 Aug, 117(4), 1227 - 34
Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis; Himelblau E et al.; A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis . This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals . ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake . Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1 . When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency . CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent . In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence . In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30% . In excised leaves and whole plants treated with high levels of exogenous CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.

J Cell Biol, 1998 Aug 10, 142(3), 723 - 33
Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein; Goode BL et al.; Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast . This protein, which we named twinfilin, is composed of two cofilin-like regions . In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins . Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers . Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization . In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches . Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches . A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments . Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.

J Cell Biol, 1998 Aug 10, 142(3), 665 - 81
A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast; Seaman MN et al.; We have recently characterized three yeast gene products (Vps35p, Vps29p, and Vps30p) as candidate components of the sorting machinery required for the endosome-to-Golgi retrieval of the vacuolar protein sorting receptor Vps10p (Seaman, M.N.J., E.G . Marcusson, J.-L . Cereghino, and S.D . Emr . 1997 . J . Cell Biol . 137:79-92) . By genetic and biochemical means we now show that Vps35p and Vps29p interact and form part of a multimeric membrane-associated complex that also contains Vps26p, Vps17p, and Vps5p . This complex, designated here as the retromer complex, assembles from two distinct subcomplexes comprising (a) Vps35p, Vps29p, and Vps26p; and (b) Vps5p and Vps17p . Density gradient fractionation of Golgi/endosomal/vesicular membranes reveals that Vps35p cofractionates with Vps5p/Vps17p in a vesicle-enriched dense membrane fraction . Furthermore, gel filtration analysis indicates that Vps35p and Vps5p are present on a population of vesicles and tubules slightly larger than COPI/coatomer-coated vesicles . We also show by immunogold EM that Vps5p is localized to discrete regions at the rims of the prevacuolar endosome where vesicles appear to be budding . Size fractionation of cytosolic and recombinant Vps5p reveals that Vps5p can self-assemble in vitro, suggesting that Vps5p may provide the mechanical impetus to drive vesicle formation . Based on these findings we propose a model in which Vps35p/Vps29p/Vps26p function to select cargo for retrieval, and Vps5p/Vps17p assemble onto the membrane to promote vesicle formation . Conservation of the yeast retromer complex components in higher eukaryotes suggests an important general role for this complex in endosome-to-Golgi retrieval.

J Cell Biol, 1998 Aug 10, 142(3), 651 - 63
Retrograde traffic out of the yeast vacuole to the TGN occurs via the prevacuolar/endosomal compartment; Bryant NJ et al.; A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae . To date, two intracellular routes from the TGN to the vacuole have been identified . Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle . Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid-containing sorting motif . In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment . In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step . Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.

J Cell Biol, 1998 Aug 10, 142(3), 635 - 49
Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing; Tam A et al.; Maturation of the Saccharomyces cerevisiae a-factor precursor involves COOH-terminal CAAX processing (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) followed by cleavage of an NH2-terminal extension (two sequential proteolytic processing steps) . The aim of this study is to clarify the precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor . We demonstrated previously that Ste24p is necessary for the first NH2-terminal processing step by analysis of radiolabeled a-factor intermediates in vivo (Fujimura-Kamada, K., F.J . Nouvet, and S . Michaelis . 1997 . J . Cell Biol . 136:271-285) . In contrast, using an in vitro protease assay, others showed that Ste24p (Afc1p) and another gene product, Rce1p, share partial overlapping function as COOH-terminal CAAX proteases (Boyartchuk, V.L., M.N . Ashby, and J . Rine . 1997 . Science . 275:1796-1800) . Here we resolve these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at two steps of a-factor maturation using two methods . First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24Delta rce1Delta) reveals a previously undetected species (P0*) that fails to be COOH terminally processed, consistent with redundant roles for Ste24p and Rce1p in COOH-terminal CAAX processing . Whereas a-factor maturation appears relatively normal in the rce1Delta single mutant, the ste24Delta single mutant accumulates an intermediate that is correctly COOH terminally processed but is defective in cleavage of the NH2-terminal extension, demonstrating that Ste24p is also involved in NH2-terminal processing . Together, these data indicate dual roles for Ste24p and a single role for Rce1p in a-factor processing . Second, by using a novel set of ubiquitin-a-factor fusions to separate the NH2- and COOH-terminal processing events of a-factor maturation, we provide independent evidence for the dual roles of Ste24p . We also report here the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be cloned . We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24Delta rce1Delta) strain, implying that like yeast Ste24p, Hs Ste24p can mediate multiple types of proteolytic events.

Biofactors, 1998, 8(1-2), 139 - 42
Randomized clinical trial of enteral yeast-selenium supplementation in preterm infants; Bogye G et al.; There are no data in the literature on effects of supplementing infants with yeast-selenium . We therefore studied the impact of selenium-enriched yeast on the serum selenium concentration of preterm infants living in a selenium-low area (Hungary) . Twenty-eight preterm infants with a mean +/- SD birthweight of 962 +/- 129 g and a gestational age of 27 +/- 1 weeks were randomized into two groups at birth with respect to selenium supplementation . In the supplemental group (n = 14) infants received 4.8 mg yeast containing 5 microgram selenium daily with naso-gastric drip during the first 14 postnatal days . The nonsupplemented infants were used as a reference group . In the supplemented group the serum selenium concentration increased from 32.1 +/- 8.5 microgram/l to 41.5 +/- 6.5 microgram/l and in the nonsupplemented group it decreased from 25.9 +/ 6.8 microgram/l to 18.2 +/- 6.4 microgram/l within two weeks . The serum glutathione peroxidase activity increased from 2.97 +/- 0.73 U/20 microliter to 6.42 +/- 3.11 U/20 microliter in the supplemented group, and it did not change significantly (from 3.53 +/- 0.94 U/20 microliter to 3.85 +/- 0.95 U/20 microliter) in the nonsupplemented group . We did not observe any complications or side effects in connection with enteral yeast-selenium supplementation . It is concluded that selenium-enriched yeast is a safe and an effective form of short term enteral selenium supplementation for preterm infants.

Braz J Med Biol Res, 1998 Mar, 31(3), 355 - 63
Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene; Nobrega MP et al.; The mutants of Saccharomyces cerevisiae assigned to complementation group G199 are deficient in mitochondrial respiration and lack a functional cytochrome oxidase complex . Recombinant plasmids capable of restoring respiration were cloned by transformation of mutants of this group with a yeast genomic library . Sequencing indicated that a 2.1-kb subclone encompasses the very end (last 11 amino acids) of the PET111 gene, the COX7 gene and a new gene (YMR255W) of unknown function that potentially codes for a polypeptide of 188 amino acids (about 21.5 kDa) without significant homology to any known protein . We have shown that the respiratory defect corresponding to group G199 is complemented by plasmids carrying only the COX7 gene . The gene YMR255W was inactivated by one-step gene replacement and the disrupted strain was viable and unaffected in its ability to grow in a variety of different test media such as minimal or complete media using eight distinct carbon sources at three pH values and temperatures . Inactivation of this gene also did not affect mating or sporulation.

Reprod Nutr Dev, 1998 May-Jun, 38(3), 275 - 80
Fate of Levucell SC I-1077 yeast additive during digestive transit in lambs; Durand-Chaucheyras F et al.; The fate of a live yeast strain, which was used as a feed additive for ruminants (Levucell SC I-1077), was studied during digestive transit in two gnotoxenic lambs reared in a sterile isolator . The number of live yeast cells were counted in the rumen and in faeces after a single administration or a daily feeding of 100 mg of Levucell SC . If the supplement was not renewed, the live yeast cells persisted in the rumen for approximately 30 h at a level close to the initial value . They were then gradually cleared . They began to be excreted with the faeces approximately 8 h after their consumption and were no longer detected after 102 h . Yeast additives did not colonize the rumen . As 17 to 34% of yeast cells remained alive during their transit through the digestive tract, their effect might extend beyond the rumen the post-ruminal compartments.

Plant Cell Physiol, 1998 Jun, 39(6), 590 - 9
Isolation of a tobacco cDNA encoding Sar1 GTPase and analysis of its dominant mutations in vesicular traffic using a yeast complementation system; Takeuchi M et al.; The cDNA clone of NtSAR1, a gene encoding the small GTPase Sar1p which is essential for vesicle formation from the endoplasmic reticulum (ER) membrane in yeast, has been isolated from Nicotiana tabacum BY-2 cells . NtSAR1 as well as AtSAR1 cDNA isolated from Arabidopsis thaliana {d'Enfert et al . (1992) EMBO J . 11: 4205} could complement the lethality of the disruption of SAR1 in yeast cells in a temperature-sensitive fashion . They also suppressed yeast sec12 and sec16 temperature-sensitive mutations as yeast SAR1 does . Using this complementation system, we analyzed the phenotypes of several mutations in plant SAR1 cDNAs in yeast cells . The expression of NtSAR1 H74L and AtSAR1 N129I showed dominant negative effect in growth over the wild-type SAR1, which was accompanied by the arrest of ER-to-Golgi transport . Such dominant mutations will be useful to analyze the role of membrane trafficking in plant cells, if their expression can be regulated conditionally.

J Biol Chem, 1998 Aug 14, 273(33), 21054 - 60
Novel localization of a Na+/H+ exchanger in a late endosomal compartment of yeast . Implications for vacuole biogenesis; Nass R et al.; Na+/H+ exchangers catalyze the electrically silent countertransport of Na+ and H+, controlling the transmembrane movement of salt, water, and acid-base equivalents, and are therefore critical for Na+ tolerance, cell volume control, and pH regulation . In contrast to numerous well studied plasma membrane isoforms (NHE1-4), much less is known about intracellular Na+/H+ exchangers, and thus far no vertebrate isoform has been shown to have an exclusively endosomal distribution . In this context, we show that the yeast NHE homologue, Nhx1 (Nass, R., Cunningham, K . W., and Rao, R . (1997) J . Biol . Chem . 272, 26145-26152), localizes uniquely to prevacuolar compartments, equivalent to late endosomes of animal cells . In living yeast, we show that these compartments closely abut the vacuolar membrane in a striking bipolar distribution, suggesting that vacuole biogenesis occurs at distinct sites . Nhx1 is the founding member of a newly emergent cluster of exchanger homologues, from yeasts, worms, and humans that may share a common intracellular localization . By compartmentalizing Na+, intracellular exchangers play an important role in halotolerance; furthermore, we hypothesize that salt and water movement into vesicles may regulate vesicle volume and pH and thus contribute to vacuole biogenesis.

Electrophoresis, 1998 Jun, 19(8-9), 1239 - 46
Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places; van Brabant AJ et al.; DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks . We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes . The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid . A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments . Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

Mol Biol Cell, 1998 Aug, 9(8), 2201 - 16
A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex; Nguyen T et al.; The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center . Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle . The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate . We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110-221 allele, which encodes mutations in the amino terminus . The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast . The spc98-63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p . In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus . Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent . The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other . Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p . By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98-63 spc110-221 cells is caused by the failure of Spc98-63p to interact with Spc110-221p . In contrast, the lethal phenotype in spc97-62 spc110-221 cells can be attributed to a decreased interaction between Spc97-62p and Spc98p . Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB . Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

Mol Biol Cell, 1998 Aug, 9(8), 2107 - 23
Mutational effect of fission yeast polalpha on cell cycle events; Bhaumik D et al.; Polalpha is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis . In this study, we investigated the cell cycle responses induced by mutations in polalpha+ . Germinating spores carrying either a deletion of polalpha+ (polalphaDelta) or a structurally intact but catalytically dead polalpha mutation proceed to inappropriate mitosis with no DNA synthesis . This suggests that the catalytic function, and not the physical presence of Polalpha, is required to generate the signal that prevents the cells from entering mitosis prematurely . Cells with a polalphats allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polalphats in cdc20 (polepsilon mutant) background arrested with a cdc phenoytpe, not a polalphats-like phenotype . At 25 degrees C, replication perturbation caused by polalphats alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability . At 36 degrees C, replication disruption caused by polalphats alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis . Together, our results indicate that the initiation DNA structure synthesized by Polalpha is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polalphats mutations induce differential downstream kinase responses.

Mol Biol Cell, 1998 Aug, 9(8), 2051 - 68
The kinesin-related proteins, Kip2p and Kip3p, function differently in nuclear migration in yeast; Miller RK et al.; The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated . Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants . By indirect immunofluorescence, the cytoplasmic microtubules in kip2Delta were consistently short or absent throughout the cell cycle . In contrast, in kip3Delta strains, the cytoplasmic microtubules were significantly longer than wild type at telophase . Furthermore, in the kip3Delta cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud . Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules . Genetic analysis demonstrated that the kip2Delta kar9Delta double mutants were synthetically lethal, whereas kip3Delta kar9Delta double mutants were viable . Conversely, kip3Delta dhc1Delta double mutants were synthetically lethal, whereas kip2Delta dhc1Delta double mutants were viable . We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms . We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration . Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.

Mol Biol Cell, 1998 Aug, 9(8), 2025 - 36
ADP-Ribosylation factors do not activate yeast phospholipase Ds but are required for sporulation; Rudge SA et al.; ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2 . The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate . arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores . The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation . Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated . This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants . Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD . These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities . The implications of these results are discussed with respect to current models of Arf signaling.

Biochemistry, 1998 Aug 4, 37(31), 10861 - 5
Detection of GTP and Pi in wild-type and mutated yeast microtubules: implications for the role of the GTP/GDP-Pi cap in microtubule dynamics; Dougherty CA et al.; Microtubule dynamics are believed to be controlled by a stabilizing cap of tubulin dimers at microtubule ends that contain either GTP or GDP and Pi in the exchangeable nucleotide site (E-site) of the beta-subunit . However, it has been difficult to obtain convincing evidence to support this hypothesis because the quantity of GTP and Pi in the E-site of assembled brain tubulin (the tubulin used in most studies thus far) is extremely low . In this study, we have measured the amount of GTP and Pi in the E-site of wild-type and mutated yeast assembled tubulins . In contrast to brain microtubules, 6% of the tubulin in a wild-type yeast microtubule contains a combination of E-site GTP and Pi . This result indicates that GTP hydrolysis and Pi release are not coupled to dimer addition to the end of the microtubule and supports the hypothesis that microtubules contain a cap of tubulin dimers with GTP or Pi in their E-sites . In addition, we have measured the E-site content of GTP and Pi in microtubules assembled from two yeast tubulins that had been mutated at residues T107 and T143 in beta-tubulin, sites thought to interact with the nucleotide bound in the E-site . Previous studies have shown that microtubules containing these mutated tubulins have modified dynamic behavior in vitro . The results from these experiments indicate that the GTP or GDP-Pi cap model does not adequately explain yeast microtubule dynamic behavior.

Proc Natl Acad Sci U S A, 1998 Aug 4, 95(16), 9161 - 6
Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast; Hochstenbach F et al.; The cell wall protects fungi against lysis and determines their cell shape . Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive . Here, we describe a fission yeast gene, ags1(+), which encodes a putative alpha-glucan synthase . In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan . In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane . Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells . These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis.

J Exp Med, 1998 Aug 3, 188(3), 483 - 95
Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes; Green LL et al.; The relationship between variable (V) gene complexity and the efficiency of B cell development was studied in strains of mice deficient in mouse antibody production and engineered with yeast artificial chromosomes (YACs) containing different sized fragments of the human heavy (H) chain and kappa light (L) chain loci . Each of the two H and the two kappa chain fragments encompasses, in germline configuration, the same core variable and constant regions but contains different numbers of unique VH (5 versus 66) or Vkappa genes (3 versus 32) . Although each of these YACs was able to substitute for its respective inactivated murine counterpart to induce B cell development and to support production of human immunoglobulins (Igs), major differences in the efficiency of B cell development were detected . Whereas the YACs with great V gene complexity restored efficient development throughout all the different recombination and expression stages, the YACs with limited V gene repertoire exhibited inefficient differentiation with significant blocks at critical stages of B cell development in the bone marrow and peripheral lymphoid tissues . Our analysis identified four key checkpoints regulated by VH and Vkappa gene complexity: (a) production of functional mu chains at the transition from the pre B-I to the pre B-II stage; (b) productive VkappaJkappa recombination at the small pre B-II stage; (c) formation of surface Ig molecules through pairing of mu chains with L chains; and (d) maturation of B cells . These findings demonstrate that V gene complexity is essential not only for production of a diverse repertoire of antigen-specific antibodies but also for efficient development of the B cell lineage.

EMBO J, 1998 Aug 3, 17(15), 4257 - 65
The pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast; Piper P et al.; Exposure of Saccharomyces cerevisiae to sorbic acid strongly induces two plasma membrane proteins, one of which is identified in this study as the ATP-binding cassette (ABC) transporter Pdr12 . In the absence of weak acid stress, yeast cells grown at pH 7.0 express extremely low Pdr12 levels . However, sorbate treatment causes a dramatic induction of Pdr12 in the plasma membrane . Pdr12 is essential for the adaptation of yeast to growth under weak acid stress, since Deltapdr12 mutants are hypersensitive at low pH to the food preservatives sorbic, benzoic and propionic acids, as well as high acetate levels . Moreover, active benzoate efflux is severely impaired in Deltapdr12 cells . Hence, Pdr12 confers weak acid resistance by mediating energy-dependent extrusion of water-soluble carboxylate anions . The normal physiological function of Pdr12 is perhaps to protect against the potential toxicity of weak organic acids secreted by competitor organisms, acids that will accumulate to inhibitory levels in cells at low pH . This is the first demonstration that regulated expression of a eukaryotic ABC transporter mediates weak organic acid resistance development, the cause of widespread food spoilage by yeasts . The data also have important biotechnological implications, as they suggest that the inhibition of this transporter could be a strategy for preventing food spoilage.

J Biochem (Tokyo), 1998 Aug, 124(2), 326 - 31
A novel 66-kDa stress protein, p66, associated with the process of cyst formation of Physarum polycephalum is a Physarum homologue of a yeast actin-interacting protein, AIP1; Matsumoto S et al.; When exposed to various stresses including heat shock, myxoamoebae, growing haploid cells of Physarum polycephalum, show marked morphological changes and consequently become disk-shaped microcysts . We have found that p66 is induced exclusively in the course of microcyst formation and has an actin-binding activity . In this study, we purified p66 to homogeneity and isolated a p66 cDNA . The deduced protein sequence contained 601 amino acids and showed 31% identity to a yeast actin-interacting protein, AIP1 . Northern blot analysis revealed that the amount of p66 mRNA was significantly increased by heat shock in myxoamoebae but not in plasmodia . Thus, p66 seems to be a developmentally-expressed stress protein which regulates the rearrangement of actin organization during microcyst formation in P . polycephalum.

Biochim Biophys Acta, 1998 Jul 23, 1381(2), 234 - 40
Dielectric behavior of budding yeast in cell separation; Asami K et al.; Dielectric behavior of budding yeast in cell separation was studied by comparing two types of temperature-sensitive cell division cycle mutants that arrest before and after cell separation at the restrictive temperature . Single spherical cells before budding but after cell separation showed one main dielectric dispersion (centered at about 1 MHz) and one additional dielectric dispersion (at about 20 MHz), which were the Maxwell-Wagner dispersions due to the plasma membrane and due to both of the cell wall and vacuole, respectively . With cells that accumulated as doublets at a point immediately before cell separation, one more dielectric dispersion appeared around 200 kHz in addition to two dielectric dispersions similar to those found for the single cells without bud . The difference in dielectric behavior between the two types of cells might be mainly attributed to the difference in cell shape, which was theoretically examined using non-spherical cell models .

Mutat Res, 1998 May 25, 400(1-2), 45 - 58
Yeast ARMs (DNA at-risk motifs) can reveal sources of genome instability; Gordenin DA et al.; The genomes of all organisms contain an abundance of DNA repeats which are at-risk for causing genetic change . We have used the yeast Saccharomyces cerevisiae to investigate various repeat categories in order to understand their potential for causing genomic instability and the role of DNA metabolism factors . Several types of repeats can increase enormously the likelihood of genetic changes such as mutation or recombination when present either in wild type or mutants defective in replication or repair . Specifically, we have investigated inverted repeats, homonucleotide runs, and short distant repeats and the consequences of various DNA metabolism mutants . Because the at-risk motifs (ARMs) that we characterized are sensitive indicators, we have found that they are useful tools to reveal new genetic factors affecting genome stability as well as to distinguish subtle differences between alleles .

J Biol Chem, 1998 Aug 7, 273(32), 20463 - 72
Evidence that partial unwrapping of DNA from nucleosomes facilitates the binding of heat shock factor following DNA replication in yeast; Geraghty DS et al.; In the yeast Saccharomyces cerevisiae, heat shock transcription factor (HSF) binds heat shock element (HSE) DNA shortly after DNA replication, independently of its activation by heat shock . To determine if HSF binding occurs before newly replicated DNA is packaged into nucleosomes, we inserted an HSE into a DNA segment that normally forms a positioned nucleosome in vivo . Transcription from constructs designed to create steric competition between binding of HSF and histone H2A-H2B dimers was generally poor, suggesting that nucleosome assembly precedes and inhibits HSF binding . However, one such construct was as transcriptionally active as a nucleosome-free control . Structural analyses suggested that approximately 40 base pairs of DNA, including the HSE, had unwrapped from the 3' edge of the histone octamer, allowing HSF to bind; approximately 100 base pairs remained in association with the histone octamer, with the same translational and rotational orientation as was seen for the poorly transcribed constructs . Modeling studies suggest that the active and inactive constructs differ from one another in the ease with which the HSE and flanking sequences can adopt the curvature needed to form a stable nucleosome . These differences may influence the probability of DNA unwrapping from already assembled nucleosomes and the subsequent binding of HSF.

J Biol Chem, 1998 Aug 7, 273(32), 20404 - 16
cpc-3, the Neurospora crassa homologue of yeast GCN2, encodes a polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains required for general amino acid control; Sattlegger E et al.; Based on characteristic amino acid sequences of kinases that phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha kinases), degenerate oligonucleotide primers were constructed and used to polymerase chain reaction-amplify from genomic DNA of Neurospora crassa a sequence encoding part of a putative protein kinase . With this sequence an open reading frame was identified encoding a predicted polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains . The 1646 amino acid sequence of this gene, called cpc-3, showed 35% positional identity over almost the entire sequence with GCN2 of yeast, which stimulates translation of the transcriptional activator of amino acid biosynthetic genes encoded by GCN4 . Strains disrupted for cpc-3 were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes in amino acid-deprived cells . The cpc-3 mutation did not affect the ability to up-regulate mRNA levels of cpc-1, encoding the GCN4 homologue and transcriptional activator of amino acid biosynthetic genes in N . crassa, but the mutation abolished the dramatic increase of CPC1 protein level in response to amino acid deprivation . These findings suggest that cpc-3 is the functional homologue of GCN2, being required for increased translation of cpc-1 mRNA in amino acid-starved cells.

J Biol Chem, 1998 Aug 7, 273(32), 20363 - 71
Yeast positive-stranded virus-like RNA replicons . 20 S and 23 S RNA terminal nucleotide sequences and 3' end secondary structures resemble those of RNA coliphages; Rodriguez-Cousino N et al.; Saccharomyces cerevisiae strains carry single-stranded RNAs called 20 S RNA and 23 S RNA . These RNAs and their double-stranded counterparts, W and T dsRNAs, have been cloned and sequenced . A few nucleotides at both ends, however, remained unknown . These RNAs do not encode coat proteins but their own RNA-dependent RNA polymerases that share a high degree of conservation to each other . The polymerases are also similar to the replicases of RNA coliphages, such as Qbeta . Here we have determined the nucleotide sequences of W and T dsRNAs at both ends using reverse transcriptase polymerase chain reaction-generated cDNA clones . We confirmed the terminal sequences by primer-extension and RNase protection experiments . Furthermore, these analyses demonstrated that W and T dsRNAs and their single-stranded RNA counterparts (i) are linear molecules, (ii) have identical nucleotide sequences at their ends, and (iii) have no poly(A) tails at their 3' ends . Both 20 S and 23 S RNAs have GGGGC at the 5' ends and the complementary 5-nucleotides sequence, GCCCC-OH, at their 3' ends . S1 and V1 secondary structure-mapping of the 3' ends of 20 S and 23 S RNAs shows the presence of a stem-loop structure that partially overlaps with the conserved 3' end sequence . Nucleotide sequences and stem-loop structures similar to those described here have been found at the 3' ends of RNA coliphages . These data, together with the similarity of the RNA-dependent RNA polymerases encoded among these RNAs and RNA coliphages, suggest that 20 S and 23 S RNAs are plus-strand single-stranded virus-like RNA replicons in yeast.

J Biol Chem, 1998 Aug 7, 273(32), 20252 - 60
Identification of active site residues in the "GyrA" half of yeast DNA topoisomerase II; Liu Q et al.; Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands . Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo . Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A . When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA . Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage . For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.

Genes Cells, 1998 May, 3(5), 307 - 19
Sequence elements that contribute to the degradation of yeast G alpha; Schauber C et al.; BACKGROUND: Gpa1 is the alpha subunit of the yeast G-protein that regulates signal transduction during mating . The stability of Galpha/Gpa1 is influenced by the ubiquitin-dependent N-end rule pathway, suggesting that the regulation of G alpha levels may be important for effective mating response and recovery . RESULTS: The G alpha sequences that confer sensitivity to degradation by the N-end rule pathway were identified . The insertion of this degradation signal (G1-Deg) into the ordinarily stable Gpa2 protein conferred proteolytic targeting . We examined G alpha degradation under different conditions and found that it was efficiently degraded in haploid and diploid cells, but was stable if it was synthesized prior to expression of the N-end rule pathway . Interestingly, a specific mutation in G alpha that is believed to promote the GTP-bound form (N388K) caused accelerated degradation . CONCLUSION: A region encompassing a putative effector-binding domain (G1-Deg) is required for G alpha degradation via the N-end rule pathway . Our studies have shown that G alpha is susceptible to proteolysis soon after synthesis . These results are in agreement with the idea that G alpha is more unstable in the GTP-bound form, which is the predominant state of monomeric/free G alpha soon after synthesis . It is likely that the signal transduced by Gbetagamma can be regulated by adjusting the levels of G alpha through proteolysis.

Protein Sci, 1998 Jul, 7(7), 1531 - 7
About the pKa of the active-site histidine in flavocytochrome b2 (yeast L-lactate dehydrogenase); Rao KS et al.; Flavocytochrome b2 or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner . When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved . During this reaction, the substrate alpha-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate . Thus, when bound to the reduced enzyme, this hydrogen is sticky . In the context of a carbanion mechanism, it resides on Nepsilon of His373, the active site base . We have shown before that a correlation between the amount of intermolecular hydrogen transfer from {2-3H} lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, kHe, for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122) . In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM . It is thus probable that exchange occurs directly with water molecules . The second-order rate constant for exchange is then 0.16 (+/-0.03) M(-1) s(-1) . Using the Eigen equation, this figure yields a pKa of 9.1+/-0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, J Biochem 67:291-295) . The mechanistic significance of these results is discussed.

Acta Univ Palacki Olomuc Fac Med, 1998, 141, 7 - 16
Signalling towards cell wall synthesis in budding yeast; Raclavsky V; The budding yeast Saccharomyces cerevisiae has long proved to be a very useful model in cell biology . Its cell morphology is established and maintained at least in part by the cell wall, a rigid but dynamic structure that affords mechanical protection . Although fungal cell walls represent an unique phenomenon, recent progress in research has shown striking parallels between yeast and mammalian cells in the area of cell morphogenesis and proliferation . Further studies promise to shed common light on the processes of cell morphogenesis including the intersections with proliferation control . This review focuses on the recent progress in this promising area in the yeast Saccharomyces cerevisiae . The process of cell wall synthesis in Saccharomyces cerevisiae was reviewed by several authors recently . Briefly, the cell wall represents a complex structure of cross-linked chitin, beta-(1,6)-d-glucan, beta-(1,3)-D-glucan and mannoproteins . Chitin and beta-(1,3)-D-glucan are synthesized by enzymatic complexes at the cell membrane and extruded into the periplasmic space, mannoproteins are synthesized along the yeast secretory pathway, and the site of beta-(1,6)-D-glucan synthesis is still unknown . The principal motif which interconnects individual cell wall constituents was recently identified by Kollar et al . The mechanisms of cross-linking of the polymers in the wall remain unknown, however . Recently, nevertheless, substantial progress has been achieved in understanding the signalling pathways which target the cell wall construction.

Curr Genet, 1998 Jul, 34(1), 21 - 9
Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication; Johnson RE et al.; The RTH1(RAD27) gene of Saccharomyces cerevisiae encodes a structure-specific endonuclease that cleaves 5'-ended single-stranded DNA at its junction with duplex DNA . Genetic and biochemical studies have indicated a role of Rth1 nuclease in the removal of RNA primers formed during DNA replication . The rth1Delta mutation confers temperature-sensitive lethality, and increases sensitivity to alkylating agents . The instability of repetitive DNA is greatly enhanced in the rth1Delta mutant . The conditional lethality of the rth1Delta mutation indicates that another nuclease can function in DNA replication in the absence of RTH1 . RAD2, a homolog of RTH1, is required for nucleotide-excision repair . Here, we examine three other homologs of RTH1/RAD2 - YEN1, EXO1, and DIN7 . Deletion of any of these genes in the rth1Delta strain has no effect on cell viability, suggesting the involvement of another, and as yet unidentified, nuclease in the maturation of Okazaki fragments . Our data also indicate that only RTH1 functions in the repair of alkylation damage . Deletions of YEN1, EXO1, DIN7, or RAD2, either singly or when combined with one another and with the rth1Delta mutation, have no effect on the rate of instability of dinucleotide repeats or on the rate of formation of large duplications in the CAN1 gene . These data provide evidence of a high degree of specificity for the role of RTH1 in DNA replication and in base-excision repair, and for the requirement of RAD2 in nucleotide-excision repair . The possibility that both Rth1 and Exo1 function in DNA mismatch repair is discussed.

J Cell Sci, 1998 Aug, 111 ( Pt 16), 2455 - 64
Escape of mitochondrial DNA to the nucleus in yme1 yeast is mediated by vacuolar-dependent turnover of abnormal mitochondrial compartments; Campbell CL et al.; Inactivation of Yme1p, a mitochondrially-localized ATP-dependent metallo-protease in the yeast Saccharomyces cerevisiae, causes a high rate of DNA escape from mitochondria to the nucleus as well as pleiotropic functional and morphological mitochondrial defects . The evidence presented here suggests that the abnormal mitochondria of a yme1 strain are degraded by the vacuole . First, electron microscopy of Yme1p-deficient strains revealed mitochondria physically associated with the vacuole via electron dense structures . Second, disruption of vacuolar function affected the frequency of mitochondrial DNA escape from yme1 and wild-type strains . Both PEP4 or PRC1 gene disruptions resulted in a lower frequency of mitochondrial DNA escape . Third, an in vivo assay that monitors vacuole-dependent turnover of the mitochondrial compartment demonstrated an increased rate of mitochondrial turnover in yme1 yeast when compared to the rate found in wild-type yeast . In this assay, vacuolar alkaline phosphatase, encoded by PHO8, was targeted to mitochondria in a strain bearing disruption to the genomic PHO8 locus . Maturation of the mitochondrially localized alkaline phosphatase pro-enzyme requires proteinase A, which is localized in the vacuole . Therefore, alkaline phosphatase activity reflects vacuole-dependent turnover of mitochondria . This assay reveals that mitochondria of a yme1 strain are taken up by the vacuole more frequently than mitochondria of an isogenic wild-type strain when these yeast are cultured in medium necessitating respiratory growth . Degradation of abnormal mitochondria is one pathway by which mitochondrial DNA escapes and migrates to the nucleus.

Radiats Biol Radioecol, 1998 May-Jun, 38(3), 451 - 5
{Rapid recovery in diploid yeast cells irradiated sequentially with fast electrons and alpha particles}; Kabakova NM et al.; In the present work, fast repair after combined irradiation by fast electrons and alpha particles was studied in diploid yeast cells Saccharomyces cerevisiae of wild-type XS 800 and its radiosensitive mutant XS 1898 . It was shown, that the fast repair significantly contribute to detected survival of the wild-type cells, while this repair is absent in XS 1898 mutant cells . An efficiency of the fast repair under combined irradiation (electrons + alpha particles), assessed on DMF of alpha particles, is lower, than that under irradiation by electrons and alpha particles separately.

Plant J, 1998 Apr, 14(1), 111 - 20
Molecular cloning and functional expression in yeast of CYP76B1, a xenobiotic-inducible 7-ethoxycoumarin O-de-ethylase from Helianthus tuberosus; Batard Y et al.; In order to obtain plant markers of chemical stress and possible tools for the bio-monitoring of pollution, a protein purification/PCR approach was used to isolate cDNAs of xenobiotic-inducible P450 oxygenases . O-dealkylation of 7-ethoxycoumarin is catalysed in Helianthus tuberosus by cytochromes P450 strongly inducible by a wide range of xenobiotics . Therefore, a 7-ethoxycoumarin O-de-ethylase (ECOD) was purified from induced tuber tissues (Batard et al., 1995) . A primer designed from an internal peptide sequence, but also corresponding to a conserved P450 haem-binding region, led to the generation of a gene-specific probe corresponding to a P450 strongly inducible by aminopyrine . Two partial and 98% identical coding sequences were isolated from a cDNA library prepared from aminopyrine-induced tuber . A full-length cDNA was reconstituted by 5'-RACE elongation . The protein deduced from this full-length sequence, with 41.1% amino acid identity to CYP76A1 and high phylogenetic relationship to other CYP76s, was termed CYP76B1 . CYP76B1 was expressed in yeast . Microsomes from the transformed yeast catalysed the NADPH-dependent O-dealkylation of 7-ethoxycoumarin . However, protein sequence as well as enzymological data indicated that CYP76B1 does not correspond to the purified ECOD protein . These results confirm previous data and demonstrate that several P450s in H . tuberosus are capable of actively catalysing the O-de-ethylation of ethoxycoumarin . Determination of the steady-state level of CYP76B1 transcripts after slicing tuber tissues and ageing them in water, alone or in the presence of various chemicals, showed that the expression of this P450 was not responsive to mechanical stress, but was strongly induced by chemical treatments . CYP76B1 thus appears to be a good potential marker of chemical stress and of environmental pollution.

Plant J, 1998 Mar, 13(6), 815 - 21
Two isoforms of plant RAD23 complement a UV-sensitive rad23 mutant in yeast; Sturm A et al.; DNA repair by nucleotide excision (NER) has been demonstrated in plant cells at the biochemical level but until now none of the molecular components of the plant NER complex has been identified . In this paper, the cloning and characterization of two isoforms of RAD23 from carrot (Daucus carota L.) are reported . It has been suggested that RAD23 in yeast is an assembly factor of the NER complex required for transcription-coupled repair as well as efficient overall genome repair . A functional assay demonstrated that both plant homologues complement the UV-sensitive phenotype of a rad23 deletion mutant of yeast . This result suggests that homologous polypeptides may catalyse NER in plants and yeast and, possibly, by a similar mechanism.

Plant J, 1998 Feb, 13(4), 465 - 73
The Arabidopsis thaliana cDNAs coding for eIF4E and eIF(iso)4E are not functionally equivalent for yeast complementation and are differentially expressed during plant development; Rodriguez CM et al.; Two cDNAs (At.EIF4E1 and At.EIF4E2) encoding, respectively, the eukaryotic initiation factors eIF4E and eIF(iso)4E of Arabidopsis thaliana were isolated by complementation of a Saccharomyces cerevisiae conditional mutant . The deduced amino acid sequences of the proteins are homologous to those from monocotyledonous plants, yeast and mammals . The corresponding genes were identified in YAC clones mapping to chromosome IV (At.EIF4E1) and to chromosome V (At.EIF4E2) . The yeast strain complemented by At.EIF4E2 grew poorly compared with an isogenic strain expressing At.EIF4E1 . Northern and in situ hybridization analysis show that both Arabidopsis At.EIF4E1 and At.EIF4E2 mRNAs are differentially accumulated in plant tissues . The At.EIF4E1 mRNA is expressed in all tissues except in the cells of the specialization zone of the roots; the At.EIF4E2 mRNA is particularly abundant in floral organs and in young developing tissues . This work further demonstrates an association between a high level of EIF4E mRNAs and cell proliferation and suggests that the plant eIF4E isoforms may have distinct functions in cell development and metabolism.

Biochem Mol Biol Int, 1998 Jul, 45(3), 475 - 87
Comparison of inactivation and conformational changes of native and apo yeast alcohol dehydrogenase during thermal denaturation; Yang Y et al.; The conformational changes of native and apo yeast alcohol dehydrogenase during thermal denaturation have been followed by fluorescence emission and circular dichroism spectra . A comparison of inactivation and conformational changes during thermal denaturation shows that for the native enzyme and for the apo-I YADH which has the conformational zinc removed, the extent of inactivation was larger than the extent of conformational changes at the same temperature . This result supported the suggestion by Tsou (Trends Biochem . Sci . 1986, 11, 427-429: Science 1993, 262, 380-381) that the enzyme active site is more flexible . The results also show that apo-I YADH without the conformational zinc was more easily inactivated with increasing incubation temperature, indicating that the stability of the apo-I YADH decreased . Kinetic analysis suggest that the substrate does not provide any protective effect during thermal inactivation of native and apo-I YADH.

Z Naturforsch {C}, 1998 May-Jun, 53(5-6), 347 - 51
Yeast permeabilization as a tool for measurement of in situ enzyme activity: localization of alkaline phosphatase; Spasova D et al.; The biochemical and ultracytochemical localization of alkaline phosphatases in permeabilized cells of Saccharomyces cerevisiae 257 has been studied . The treatment with non-ionic surfactant Triton X-100 allows the penetration of the substrate into intact yeast cells and thus provides detailed detection of the enzyme activity in ultracytochemical studies.

J Cell Biol, 1998 Jul 27, 142(2), 443 - 55
Iqg1p, a yeast homologue of the mammalian IQGAPs, mediates cdc42p effects on the actin cytoskeleton; Osman MA et al.; The Rho-type GTPase Cdc42p has been implicated in diverse cellular functions including cell shape, cell motility, and cytokinesis, all of which involve the reorganization of the actin cytoskeleton . Targets of Cdc42p that interface the actin cytoskeleton are likely candidates for mediating cellular activities . In this report, we identify and characterize a yeast homologue for the mammalian IQGAP, a cytoskeletal target for Cdc42p . The yeast IQGAP homologue, designated Iqg1p, displays a two-hybrid interaction with activated Cdc42p and coimmunoprecipitates with actin filaments . Deletion of IQG1 results in a temperature-sensitive lethality and causes aberrant morphologies including elongated and round multinucleated cells . This together with its localization at the mother-bud neck, suggest that Iqg1p promotes budding and cytokinesis . At restrictive temperatures, the vacuoles of the mutant cells enlarge and vesicles accumulate in the bud . Interestingly, Iqg1p shows two-hybrid interactions with the ankyrin repeat-containing protein, Akr1p (Kao, L.-R., J . Peterson, J . Ruiru, L . Bender, and A . Bender . 1996 . Mol . Cell . Biol . 16:168-178), which inhibits pheromone signaling and appears to promote cytokinesis and/or trafficking . We also show two-hybrid interactions between Iqg1p and Afr1p, a septin-binding protein involved in projection formation (Konopka, J.B., C . DeMattei, and C . Davis . 1995 . Mol . Cell . Biol . 15:723-730) . We propose that Iqg1p acts as a scaffold to recruit and localize a protein complex involved in actin-based cellular functions and thus mediates the regulatory effects of Cdc42p on the actin cytoskeleton.

J Cell Biol, 1998 Jul 27, 142(2), 403 - 20
Pex20p of the yeast Yarrowia lipolytica is required for the oligomerization of thiolase in the cytosol and for its targeting to the peroxisome; Titorenko VI et al.; Pex mutants are defective in peroxisome assembly . In the pex20-1 mutant strain of the yeast Yarrowia lipolytica, the peroxisomal matrix protein thiolase is mislocalized exclusively to the cytosol, whereas the import of other peroxisomal proteins is unaffected . The PEX20 gene was isolated by functional complementation of the pex20-1 strain and encodes a protein, Pex20p, of 424 amino acids (47,274 D) . Despite its role in the peroxisomal import of thiolase, which is targeted by an amino-terminal peroxisomal targeting signal-2 (PTS2), Pex20p does not exhibit homology to Pex7p, which acts as the PTS2 receptor . Pex20p is mostly cytosolic, whereas 4-8% is associated with high-speed (200,000 g) pelletable peroxisomes . In the wild-type strain, all newly synthesized thiolase is associated with Pex20p in a heterotetrameric complex composed of two polypeptide chains of each protein . This association is independent of PTS2 . Pex20p is required for both the oligomerization of thiolase in the cytosol and its targeting to the peroxisome . Our data suggest that monomeric Pex20p binds newly synthesized monomeric thiolase in the cytosol and promotes the formation of a heterotetrameric complex of these two proteins, which could further bind to the peroxisomal membrane . Translocation of the thiolase homodimer into the peroxisomal matrix would release Pex20p monomers back to the cytosol, thereby permitting a new cycle of binding-oligomerization-targeting-release for Pex20p and thiolase.

J Biomed Sci, 1998, 5(3), 203 - 10
Expression of HIV-1 nef in yeast causes membrane perturbation and release of the myristylated Nef protein; Macreadie IG et al.; The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial . During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet . This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies . Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies . These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.

J Biol Chem, 1998 Jul 31, 273(31), 19884 - 91
Genetic tests of the role of Abf1p in driving transcription of the yeast TATA box bindng protein-encoding gene, SPT15; Schroeder SC et al.; In this report we describe studies which utilized yeast strains bearing gain and loss of function alleles of ABF1 in order to attempt to directly implicate Abf1p in modulating transcription of the TBP-encoding gene, SPT15, in vivo . We found that overexpression of Abf1p in a yeast cell increased transcription of the TBP-encoding gene and that this stimulation depended upon the exact sequence of the Abf1p binding site (ABF1) present in the gene . Further, in a yeast strain expressing a temperature sensitive form of Abf1p, occupancy of the chromosomal ABF1 site in the TBP-encoding gene was immediately lost following a temperature shift . Both results suggest that Abf1p drives transcription of the TBP-encoding gene . Surprisingly though we found that continuous ABF1 cis-element occupancy by Abf1p was not acutely required for normal levels of transcription of either the TBP-encoding gene or other "Abf1p-driven" genes tested . We propose a model to explain these results and suggest mechanisms by which Abf1p could activate gene transcription.

J Biol Chem, 1998 Jul 31, 273(31), 19443 - 52
Mutations in the yeast proteasome beta-type subunit Pre3 uncover position-dependent effects on proteasomal peptidase activity and in vivo function; Gueckel R et al.; Proteasomes are highly complex proteases responsible for selective protein degradation in the eukaryotic cell . 26 S proteasomes consist of two regulatory 19 S cap complexes and the 20 S proteasome, which acts as the proteolytic core module . We isolated six mutants of the yeast Saccharomyces cerevisiae containing mutations in the 20 S proteasome beta-type subunit Pre3 . Three mutations (pre3-2, pre3-3, and pre3-5) which reside at the active site cleft of the Pre3 subunit solely caused reduction of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in protein degradation nor to any other phenotype . However, the pre3-2 mutation strengthened phenotypes induced by other 20 S proteasomal mutations, indicating that the peptidylglutamyl peptide-hydrolyzing activity has to fulfill some rescue functions . The other three mutations (pre3-1, pre3-4, and pre3-6) are located at diverse sites of the Pre3 protein and caused multiple defects in proteasomal peptide cleaving activities . pre3-1 and pre3-6 mutants exhibited significant defects in proteasomal protein degradation; they accumulated ubiquitinated proteins and stabilized defined substrate proteins as, e.g . fructose-1,6-bisphosphatase . In addition, pre3-1 and pre3-6 mutant cells exhibited pleiotropic phenotypes as temperature sensitivity and cell cycle-related effects.

Biochem J, 1998 Aug 1, 333 ( Pt 3), 839 - 45
Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass; Foley V et al.; Two similar glutathione S-transferases (GSTs), which do not bind to glutathione- or S-hexylglutathione-agarose affinity resins, have been purified from the yeast Yarrowia lipolytica . An approx . 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography . The native molecular mass of both proteins was estimated as approx . 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis . SDS/PAGE indicated a subunit mass of 50 kDa . Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers . Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar . The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium {Dowd, Buckley and Sheehan (1997) Biochem . J . 324, 243-248} . It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

Yeast, 1998 Jun 15, 14(8), 773 - 81
N-glycosylation by transfer of GlcNAc2 from dolichol-PP-GlcNAc2 to the protein moiety of the major yeast exoglucanase; Cueva R et al.; Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated . When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme . As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc . Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N4-N-acetyl-beta-glucosamine asparagine amidase . These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PP-GlcNAc2 to one or both sequons of yeast Exg . Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum.

Extremophiles, 1998 Jan, 2(1), 41 - 9
Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica; Deegenaars ML et al.; The response to heat stress in six yeast species isolated from Antarctica was examined . The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20 degrees C, and the other psychrotrophic, capable of growth at temperatures above 20 degrees C . In addition to species--specific heat shock prote in (hsp) profiles, a heat shock (15 degrees C-25 degrees C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M . frigida, and M . gelida, but not evident in Leucosporidium antarcticum . Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90 . Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined . In the psychrotrophic yeast, Leucosporidium fellii and L . scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed . In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted . Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species . A 10 Celsius degree increase in temperature above the growth temperature (15 degrees C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance . On the other hand, in psychrotrophic yeast grown at 25 degrees C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance . Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation . It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles.

Proc R Soc Lond B Biol Sci, 1998 Jun 7, 265(1400), 1017 - 23
The effect of sex on adaptation to high temperature in heterozygous and homozygous yeast; Greig D et al.; Most explanations for the evolutionary maintenance of sex depend on the assumption that sex produces variation by recombining parental haplotypes in the offspring . Therefore, meiosis is expected to be useful only in heterozygotes . We tested this assumption by competing sexual strains of yeast against constitutive asexuals in a hot (37 degrees C) culture for 500 generations, in either heterozygous or homozygous genetic backgrounds . We found that there was an initial cost of sex for all the sexual strains, which was indicated by a sharp increase in the proportion of asexuals after the induction of sex . The cost was larger in the heterozygotes than in the homozygotes, probably because of recombinational load . However, in two of the three heterozygote backgrounds, after the initial success of the asexuals, the remaining sexuals eventually drove them out of the population . These two heterozygotes also suffered the largest initial cost of sex . In the other heterozygote and in the three homozygote backgrounds it appeared to be a matter of chance whether sexuals or asexuals won . The average relative fitness increased in all the strains, but the increase was largest in the two strains that showed both the clearest advantage and the largest cost of sex . We conclude that these results are consistent with the traditional view that sex has a short-term cost but a long-term benefit.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 13 - 9
The uvsI gene of Aspergillus nidulans required for UV-mutagenesis encodes a homolog to REV3, a subunit of the DNA polymerase zeta of yeast involved in translesion DNA synthesis; Han KY et al.; Defects in the uvsI gene of Aspergillus nidulans resulted in high UV sensitivity and reductions of spontaneous and UV-induced reversion of certain alleles, uvsl;uvsA double mutants exhibited high methyl methane sulfonate (MMS)-sensitivity in contrast to the slight sensitivity of the component single mutants . Using such a double mutant as recipient, a clone complementing uvsI501 has been isolated from a chromosome III specific library . The deduced amino acid sequence from the 1.1-kb sequenced region, a part of the 5.2-kb DNA fragment showing uvsI-complementing activity, had a 62% identity with REV3 of yeast . Disruptants of the cloned gene demonstrated the same level of sensitivity to UV light as uvsI and failed to complement uvsI501 in heterozygous diploids.

Biochim Biophys Acta, 1998 Jul 17, 1372(2), 261 - 71
Regulation and pH-dependent expression of a bilaterally truncated yeast plasma membrane H+-ATPase; Mason AB et al.; Constitutive, chromosomal expression of yeast pma1 deletion alleles in Saccharomyces cerevisiae yielded functional, truncated forms of the plasma membrane H+-ATPase which were independently capable of supporting wild type yeast growth rates . Deletion of 27 amino-terminal residues affected neither the enzyme's activity nor its responsiveness to changes in glucose metabolism . By contrast, removal of 18 carboxy-terminal amino acids produced an enzyme with a Vmax that was relatively insensitive to glucose-dependent metabolic status and with a Km that was significantly lower than that of the wild type enzyme . These effects were exaggerated when the amino- and carboxy-terminal deletions were combined in a bilaterally truncated H+-ATPase, suggesting that the amino terminus may have a subtle role in modulating ATPase activity . In pma1DeltaDelta cells cultured at pH 6, plasma membrane H+-ATPase levels were much lower than those in cells expressing a wild type ATPase . Increased expression levels could be achieved by growing the pma1DeltaDelta mutant at pH 3, a result that was at least partially due to a sustained, elevated transcription of pma1DeltaDelta mRNA . Our observations suggest that intracellular proton balance can be maintained by regulation of the activity and/or quantity of H+-ATPase in the plasma membrane.

Biochem Biophys Res Commun, 1998 Jul 20, 248(2), 240 - 4
Identification and cloning of two novel allergens from the lipophilic yeast, Malassezia furfur; Yasueda H et al.; Two novel allergens, designated Mal f 2 and Mal f 3 according to the WHO/IUIS Allergen Nomenclature Subcommittee recommendation, were isolated from the lipophilic yeast Malassezia furfur cell extracts and the genes coding for those were cloned . Mal f 2 and Mal f 3 had apparent molecular weights of 21 kDa and 20 kDa, respectively, on SDS-PAGE under reducing conditions . The identified cDNA clone of Mal f 2 encoded an open reading frame of 177 amino acid residues . Fifty-one percent identity was found between the Mal f 2 and Mal f 3 sequences . Comparison of the Mal f 2 and Mal f 3 sequences with known protein sequences revealed that they had sequence homology with two peroxisomal membrane proteins of Candida boidinii and an Aspergillus fumigatus allergen, Asp f 3 . In RAST, both Mal f 2- and Mal f 3-specific IgE antibodies could be detected in approximately 70 % of sera from M . furfur sensitized patients with atopic dermatitis .

Biochimie, 1998 Apr, 80(4), 321 - 4
Yeast ribosomal protein L26 is located at the ribosomal subunit interface as determined by chemical cross-linking; Villarreal J Jr et al.; Previous studies suggested that yeast ribosomal protein L26 was a candidate for the ribosomal subunit interface region . The present study used protein-protein cross-linking to identify neighboring proteins in intact 80S ribosomes of Saccharomyces cerevisiae . To facilitate identification of cross-linked pairs involving L26, 80S ribosomes were first treated with 5-iodoacetamidofluorescein to selectively label L26 . Protein cross-links were produced with dithiobis{succinimidyl} propionate or N-succinimidyl-3-{2-pyridyldithio} propionate and analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing SDS . L26 was detected under UV and its cross-linked partner, detectable after staining with Coomassie blue, was located below the diagonal and was coincident with L26 on a single vertical axis . The identity of the partner was determined by its co-migration with 60S and 40S ribosomal protein markers on two-dimensional gels . Two protein pairs involving L26 and 40S subunit proteins were identified . The finding provided experimental evidence to support that L26 is located at the ribosomal subunit interface . A model that incorporates the present findings and the published data on intra-subunit protein pairs is proposed for the yeast 80S ribosome.

Proc Natl Acad Sci U S A, 1998 Jul 21, 95(15), 8619 - 24
A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation; Murray LE et al.; Dimorphic growth of the budding yeast Saccharomyces cerevisiae is regulated by the quality of the nitrogen supply . On a preferred nitrogen source diploid cells grow as ellipsoidal cells by using a bipolar pattern of budding, whereas on a poor nitrogen source a unipolar pattern of budding is adopted, resulting in extended pseudohyphal chains of filamentous cells . Here we report that the quality of the nitrogen source is signaled by the glutamine tRNA isoform with a 5'-CUG anticodon (tRNACUG) . Mutations that alter this tRNA impair assessment of the nitrogen supply without measurably affecting protein synthesis, so that mutant cells display pseudohyphal growth even on a preferred nitrogen source . The nitrogen status for other nitrogen-responsive processes such as catabolic gene expression and sporulation also is signaled by this tRNA: mutant cells inappropriately induce the nitrogen-repressed gene CAR1 and undergo precocious sporulation in nitrogen-rich media . Therefore, in addition to its role in mRNA translation, this tRNA also transduces nitrogen signals that regulate development.

Proc Natl Acad Sci U S A, 1998 Jul 21, 95(15), 8532 - 7
A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin; Kamada S et al.; Caspase-mediated proteolysis is a critical and central element of the apoptotic process; therefore, it is important to identify the downstream molecular targets of caspases . We established a method for cloning the genes of caspase substrates by two major modifications of the yeast two-hybrid system: (i) both large and small subunits of active caspases were expressed in yeast under ADH1 promoters and the small subunit was fused to the LexA DNA-binding domain; and (ii) a point mutation was introduced that substituted serine for the active site cysteine and thereby prevented proteolytic cleavage of the substrates, possibly stabilizing the enzyme-substrate complexes in yeast . After screening a mouse embryo cDNA expression library by using the bait plasmid for caspase-3, we obtained 13 clones that encoded proteins binding to caspase-3, and showed that 10 clones including gelsolin, an actin-regulatory protein implicated in apoptosis, were cleaved by recombinant caspase-3 in vitro . Using the same bait, we also isolated human gelsolin cDNA from a human thymus cDNA expression library . We showed that human gelsolin was cleaved during Fas-mediated apoptosis in vivo and that the caspase-3 cleavage site of human gelsolin was at D352 of DQTD352G, findings consistent with previous observations on murine gelsolin . In addition, we ascribed the antiapoptotic activity of gelsolin (which we previously reported) to prevention of a step leading to cytochrome c release from the mitochondria into the cytosol . Our results indicate that this cloning method is useful for identification of the substrates of caspases and possibly also of other enzymes.

Mol Cell Biol, 1998 Aug, 18(8), 4783 - 92
Yeast Ty1 retrotransposition is stimulated by a synergistic interaction between mutations in chromatin assembly factor I and histone regulatory proteins; Qian Z et al.; A screen for host mutations which increase the rate of transposition of Ty1 and Ty2 into a chromosomal target was used to identify factors influencing retroelement transposition . The fortuitous presence of a mutation in the CAC3 gene in the strain in which this screen was undertaken enabled us to discover that double mutaions of cac3 and hir3, but neither of the two single mutations, caused a dramatic increase in the rate of retrotransposition . We further showed that this effect was not due to an increase in the overall level of Ty1 mRNA . Two subtle cac3 phenotypes, slight methyl methanesulfonate (MMS) sensitivity and reduction of telomeric silencing, were significantly enhanced in the cac3 hir3 double mutant . In addition, the growth rate of the double mutant was reduced . HIR3 belongs to a class of HIR genes that regulate the transcription of histones, while Cac3p, together with Cac1p and Cac2p, forms chromatin assembly factor I . Other combinations of mutations in cac and hir genes (cac3 hir1, cac3 hir2, and cac2 hir3) also increase Ty transposition and MMS sensitivity and reduce the growth rate . A model explaining the synergistic interaction between cac and hir mutations in terms of alterations in chromatin structure is proposed.

Mol Cell Biol, 1998 Aug, 18(8), 4597 - 604
Mapping the polarity of changes that occur in interrupted CAG repeat tracts in yeast; Maurer DJ et al.; To explore the mechanisms by which CAG trinucleotide repeat tracts undergo length changes in yeast cells, we examined the polarity of alterations with respect to an interrupting CAT trinucleotide near the center of the tract . In wild-type cells, in which most tract changes are large contractions, the changes that retain the interruption are biased toward the 3' end of the repeat tract (in reference to the direction of lagging-strand synthesis) . In rth1/rad27 mutant cells that are defective in Okazaki fragment maturation, the tract expansions are biased to the 5' end of the repeat tract, while the tract contractions that do not remove the interruption occur randomly on either side of the interruption . In msh2 mutant cells that are defective in the mismatch repair machinery, neither the small changes of one or two repeat units nor the larger contractions attributable to this mutation are biased to either side of the interruption . The results of this study are discussed in terms of the molecular paths leading to expansions and contractions of repeat tracts.

RNA, 1998 Jul, 4(7), 856 - 69
The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity; Motorin Y et al.; We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of psi34 and psi36 in the anticodon loop of the yeast minor tRNA(Ile) in vitro (Simos G et al., 1996, EMBO J 15:2270-2284) . Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA(Tyr) (anticodon GUA) . Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (psi) formation at distinct noncontiguous sites within the same tRNA molecule . The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of psi27, psi28, psi34, and psi36 in vitro, however, psi35 formation in the intron-containing tRNA(Tyr)(GUA) remains unaffected . Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA(Tyr), another site-specific tRNA:pseudouridine synthase with overlapping specificity exists . Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p . In addition, they suggest that Pus1p is implicated in modification at positions U26, U65, and U67 in vivo.

J Immunol, 1998 Jul 15, 161(2), 821 - 8
Expression screening of a yeast artificial chromosome contig refines the location of the mouse H3a minor histocompatibility antigen gene; Zuberi AR et al.; The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains . H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells . Other genes within the complex include beta2-microglobulin and H3b . A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a . This contig refines the position of many genes and anonymous loci . In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a . A novel assay was developed to determine the location of H3a within the contig . Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells . YAC-containing L cells were screened for the expression of the YAC-encoded H3a(a) Ag by using them as targets in a cell-mediated lympholysis assay with H3a(a)-specific CTLs . A single YAC carrying H3a was identified . Based on the location of this YAC within the contig, many candidate genes can be eliminated . The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3 . These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.

EMBO J, 1998 Jul 15, 17(14), 4199 - 209
Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p; Paciotti V et al.; Checkpoints prevent DNA replication or nuclear division when chromosomes are damaged . The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathway . Ddc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage . We demonstrate that Ddc1p interacts physically in vivo with Mec3p, and this interaction requires Rad17p . We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p . This suggests that Mec1p might act together with the Rad24 group of proteins at an early step of the DNA damage checkpoint response . On the other hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p . Moreover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1 . We suggest that Rad9p and Ddc1p might function in separated branches of the DNA damage checkpoint pathway, playing different roles in determining Mec1p activity and/or substrate specificity.

EMBO J, 1998 Jul 15, 17(14), 4086 - 91
The yeast galactose genetic switch is mediated by the formation of a Gal4p-Gal80p-Gal3p complex; Platt A et al.; Saccharomyces cerevisiae responds to galactose as the sole source of carbon by activating the GAL genes encoding the enzymes of the Leloir pathway . Here, we show in vitro that the switch from repressed to activated gene expression involves the interplay of three proteins {an activator (Gal4p), a repressor (Gal80p) and an inducer (Gal3p)} and two small molecules (galactose and ATP) . We also show that the galactose- and ATP-dependent interaction between Gal3p and Gal80p occurs without disruption of the Gal80p-Gal4p interaction . Thus, Gal3p-mediated activation of transcription occurs via the formation of a tripartite protein complex.

EMBO J, 1998 Jul 15, 17(14), 4004 - 17
Novel developmentally regulated phosphoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast; Kearns MA et al.; Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development . While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized . We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains . We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs . Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant . Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress . We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

Biochemistry (Mosc), 1998 Jun, 63(6), 725 - 8
Synthesis of protoporphyrin IX induced by 5-aminolevulinic acid in yeast cells in the presence of 2,2;-dipyridyl
Strakhovskaya MG, Shumarina AO, Fraikin GY, Rubin AB.
5-Aminolevulinic acid (ALA), a precursor of porphyrin synthesis, increased the production of various porphyrin compounds in Candida guilliermondii cells . Metalloporphyrins and protoporphyrin IX (PPIX) were predominantly accumulated, respectively, at ALA concentrations of 0.2-0.4 mM and at those higher than 1.5 mM . 2,2;-Dipyridyl which complexed with bivalent metals significantly increased the content of endogenous PPIX even at ALA concentrations lower than 0.5 mM . Under these conditions, the yeast sensitivity to photodynamic effect of visible light (400-600 nm) dramatically increased due to photosensitization by endogenous PPIX.

Curr Opin Chem Biol, 1998 Apr, 2(2), 216 - 21
Metal-ion regulation of gene expression in yeast; Winge DR et al.; Metal-responsive transcription factors exist in yeast to modulate expression of genes that encode proteins involved in cellular uptake of copper, iron and zinc ions . These signal transduction pathways function in the cellular regulation of the intracellular concentration of free metal ions . A second component of metal homeostasis is the regulation of metal-ion binding through protein-mediated metallation . Copper-specific chaperones exist in yeast that route copper ions to the site of biosynthesis of copper-metalloenzymes.

Ann Med, 1998 Apr, 30(2), 186 - 91
Impact and implications of yeast and human artificial chromosomes; Schlessinger D et al.; Artificial chromosomes have been developed in the last 10 years to sustain genome mapping and, more recently, to begin initiating functional studies and some approaches to gene therapy . The use of yeast artificial chromosomes (YACs) in mapping the human X chromosome is reported as an example . The requirements which have postponed the development of human artificial chromosomes have now been relatively met, and some prospects are previewed here.

Nat Struct Biol, 1998 Jul, 5(7), 551 - 5
Solution structure of a zinc domain conserved in yeast copper-regulated transcription factors; Turner RB et al.; The three dimensional structure of the N-terminal domain (residues 1-42) of the copper-responsive transcription factor Amtl from Candida glabrata has been determined by two-dimensional 1H-correlated nuclear magnetic resonance (NMR) methods . The domain contains an array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) that is conserved among a family of Cu-responsive transcription factors . The structure is unlike those of previously characterized zinc finger motifs, and consists of a three-stranded antiparallel beta-sheet with two short helical segments that project from one end of the beta-sheet . Conserved residues at positions 16, 18 and 19 form a basic patch that may be important for DNA binding.

Plant Cell Physiol, 1998 May, 39(5), 545 - 54
RMA1, an Arabidopsis thaliana gene whose cDNA suppresses the yeast sec15 mutation, encodes a novel protein with a RING finger motif and a membrane anchor; Matsuda N et al.; To identify molecules that function in the plant secretory pathway, we screened for Arabidopsis thaliana cDNA clones that complement the temperature-sensitive (ts), secretion-deficient sec15 mutation of yeast Saccharomyces cerevisiae . RMA1, one of the genes obtained in this screening, suppressed not only the ts growth of sec15 but also its secretory defect . RMA1 is not a structural homologue of SEC15 but encodes a novel 28 kDa protein with a RING finger motif and a C-terminal membrane-anchoring domain . Mutational analysis indicates that the RING finger motif of RMA1 is important for its suppression activity . In Arabidopsis plant, RMA1 is ubiquitously expressed . A search for homologous proteins in the database revealed that Arabidopsis, nematode, mouse and human possess close homologues of RMA1.

Plant Physiol, 1998 Jul, 117(3), 859 - 67
Characterization of a red beet protein homologous to the essential 36-kilodalton subunit of the yeast V-type ATPase; Bauerle C et al.; V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells . V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (Vo) and a peripherally associated catalytic sector (V1) . Several V-ATPase subunits are functionally and structurally conserved among all species examined . In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the Vo sector as well as for V1 attachment . Vma6p has been characterized as a nonintegrally associated Vo subunit . A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggest that this subunit has a conserved role in V-ATPase function . We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes . A 44-kD polypeptide cofractionated with V-ATPases upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies . The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane . The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.

Curr Biol, 1998 Jul 2, 8(14), 831 - 4
The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities; Polotnianka RM et al.; The Ku heterodimer, conserved in a wide range of eukaryotes, plays a multiplicity of roles in yeast . First, binding of Ku, which is composed of a 70 kDa (Hdf1p) and an 80 kDa (Hdf2p) subunit {1-3}, to double-strand breaks promotes non-homologous end-to-end joining of DNA {3} . Second, Ku appears to participate in DNA replication, regulating both the number of rounds of replication permissible within the cell cycle and the structure of the initiation complex {3,4} . Furthermore, mutations in HDF1 or HDF2 rapidly reduce telomeric poly (TG1-3) tract size {1-3}, hinting also at a possible telomeric function of Ku . We show here that the two subunits of the Ku heterodimer play a key role in maintaining the integrity of telomere structure . Mutations in either Ku subunit increased the single-strandedness of the telomere in a cell-cycle-independent fashion, unlike wild-type cells which form 3' poly(TG1-3) overhangs exclusively in late S phase {5} . In addition, mutations enhanced the instability of elongated telomeres to degradation and recombination . Both Ku subunits genetically interacted with the putative single-stranded telomere-binding protein Cdc13p . We propose that Ku protects the telomere against nucleases and recombinases.

Vestn Ross Akad Med Nauk, 1998, (5), 47 - 50
{Yeast-like fungi malassezia (pityrosporum): clinical and immunological aspects fo study}; Mokronosova MA et al.; The normal flora is typified by the yeast-like fungi Malassezia (Pityrosporum) . Successful attempts at treating patients with atopic and seborreic dermatitis, pityriasis versicolor, and psoriasis with antifungicides confirm the involvement of these fungi in the etiology and development of these diseases . In patients with various skin diseases, an immune response to M . furfur is specific . In those with psoriasis, it is characterized by the higher chemoatractant activity of polymorphonuclear leukocytes stimulated by a M . furfur extract and by the immune response of IgG antibodies with immunoreactive proteins having a molecular weight of 100-120 kD . Patients with atopic dermatitis show a hyperimmune IgE-mediated response to M . furfur, with its specific Th2-lymphocytes inducing the atopic cytokines (IL-4, IL-10) that stimulate allergic reactions to other allergens . Those with pityriasis versicolor had impaired keratinocytic pigment exchange due to azelainic acid produced by M . furfur . The cause of transformation of the yeast phase of M . furfur to the mycelial one is presumably to be changes in the composition of fatty acids of the sebaceous glands due to increased androgen concentrations.

Electrophoresis, 1998 Jun, 19(7), 1179 - 84
Subcellular fractionation by organelle electrophoresis: separation of phagosomes containing heat-killed yeast particles; Hasan Z et al.; Uptake of foreign material and its subsequent lysosomal degradation is an important function of macrophages . The mechanisms involved in the binding, uptake and delivery of such material to lysosomal organelles, are, however, poorly understood . Here we describe a method using organelle electrophoresis to study the uptake and trafficking of heat-killed yeast particles within murine macrophages . Such yeast particles, which were fluorescently labeled, could be readily detected in intact cells as well as in subcellular fractions . Organelle electrophoresis of a homogenate from macrophages that had internalized yeast particles resulted in the separation of yeast-containing organelles from most other subcellular membranes . In addition, this method was used to follow the kinetics of yeast particle transport within macrophages could be followed readily . Organelle electrophoresis may be a valuable tool for the analysis of phagocytosis.

Mol Cell, 1998 May, 1(6), 913 - 6
Activation of transcription in vitro by recruitment of the yeast RNA polymerase II holoenzyme; Gaudreau L et al.; It has been argued that many transcriptional activators work by "recruitment," that is, by helping the transcriptional machinery bind stably to DNA . We demonstrate here a realization of a strong prediction of this idea in an in vitro transcription reaction performed with purified yeast RNA polymerase II holoenzyme and a classical transcriptional activator . We show that the level of transcription reached by the activator working on low concentrations of holoenzyme can also be reached in the absence of activator by raising the holoenzyme concentration, and that under that condition the activator has no further stimulatory effect . We also show, in agreement with another prediction of the recruitment model, that in a reaction using a holoenzyme purified from cells bearing the "P" mutation, transcription is stimulated by a DNA-tethered peptide that binds the mutant holoenzyme component Gal11P but that lacks a classical activating region.

Mol Cell, 1998 Apr, 1(5), 685 - 96
Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80; Chu S et al.; Gametogenesis requires the successful coordination of two key processes, meiotic nuclear division and gamete morphogenesis . A central regulatory step in progression through gametogenesis occurs at the pachytene stage of meiotic prophase . We find that Ndt80 functions at pachytene of yeast gametogenesis (sporulation) to activate transcription of a set of genes required for both meiotic division (e.g., B-type cyclins) and gamete formation (e.g., SPS1) . Ectopic synthesis of Ndt80 in vegetative cells induces transcription of these genes, and recombinant Ndt80 protein binds to a conserved sequence in their upstream region . Transcription of NDT80 itself is dependent on Ime1, which activates expression of early sporulation genes . Transcription of the Ndt80-regulated gene CLB1 is mediated by the checkpoint gene RAD17 . Thus Ndt80 is a pivotal component of a transcriptional cascade programming yeast gametogenesis and may also be a target of meiotic checkpoint control.

Mol Cell, 1998 Mar, 1(4), 495 - 505
Absence of Gcn5 HAT activity defines a novel state in the opening of chromatin at the PHO5 promoter in yeast; Gregory PD et al.; Histone acetyltransferase (HAT) activity has been demonstrated for several transcriptional activators, formally connecting chromatin modification with gene regulation . However, no effect on chromatin has been demonstrated . We have investigated the role of the HAT Gcn5 at the nucleosomally regulated PHO5 promoter . Under conditions of constitutive submaximal activation (i.e., in the absence of the negative regulator Pho80), deletion of Gcn5 determines a novel randomized nucleosomal organization across the promoter and leads to a dramatic reduction in activity . Furthermore, mutation of amino acids critical for Gcn5 HAT activity is sufficient to generate this structure . This intermediate state in chromatin opening gives way to the fully open structure upon maximal induction (phosphate starvation), even in the absence of Gcn5 . Thus, Gcn5 is shown to affect directly the remodeling of chromatin in vivo.

Mol Cell, 1998 Feb, 1(3), 337 - 46
Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast; Xu Q et al.; The mammalian proapoptotic protein Bax confers a lethal phenotype when expressed in yeast . By exploiting this phenotype, we have identified a novel human Bax inhibitor, BI-1 . BI-1 is an evolutionarily conserved integral membrane protein containing multiple membrane-spanning segments and is predominantly localized to intracellular membranes, similar to Bcl-2 family proteins . Moreover, BI-1 can interact with Bcl-2 and Bcl-XL but Bax or Bak, as demonstrated by in vivo cross-linking and coimmunoprecipitation studies . When overexpressed in mammalian cells, BI-1 suppressed apoptosis included by Bax, etoposide, staurosporine, and growth factor deprivation, but not by Fas (CD95) . Conversely, BI-1 antisense induced apoptosis . BI-1 thus represents a new type of regulator of cell death pathways controlled by Bcl-2 and Bax.

J Cell Biol, 1998 Jul 13, 142(1), 39 - 49
Assembly of the yeast vacuolar H+-ATPase occurs in the endoplasmic reticulum and requires a Vma12p/Vma22p assembly complex; Graham LA et al.; Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmic reticulum (ER) . These three proteins are required for the biogenesis of a functional vacuolar ATPase (V-ATPase), but are not part of the final enzyme complex . Subcellular fractionation and chemical cross-linking studies have revealed that Vma12p and Vma22p form a stable membrane associated complex . Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase . The interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of approximately 5 min), reflecting trafficking of this V-ATPase subunit through the ER en route to the vacuolar membrane . Analysis of these protein-protein interactions in ER-blocked sec12 mutant cells indicated that the Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase out of the ER is blocked . Fractionation of solubilized membrane proteins on a density gradient revealed comigration of Vma22p and Vma12p, indicating that they form a complex even in the absence of cross-linker . Vma12p and Vma22p migrated to fractions separate from Vma21p . Loss of Vph1p caused the Vma12p/Vma22p complex to sediment to less dense fractions, consistent with association of Vma12p/ Vma22p with nascent Vph1p in ER membranes . This is the first evidence for a dedicated assembly complex in the ER required for the assembly of an integral membrane protein complex (V-ATPase) as it is transported through the secretory pathway.

J Biol Chem, 1998 Jul 17, 273(29), 18556 - 61
A linker region of the yeast zinc cluster protein leu3p specifies binding to everted repeat DNA; Mamane Y et al.; Yeast zinc cluster proteins form a major class of yeast transcriptional regulators . They usually bind as homodimers to target DNA sequences, with each monomer recognizing a CGG triplet . Orientation and spacing between the CGG triplet specifies the recognition sequence for a given zinc cluster protein . For instance, Gal4p binds to inverted CGG triplets spaced by 11 base pairs whereas Ppr1p recognizes a similar motif but with a spacing of 6 base pairs . Hap1p, another member of this family, binds to a direct repeat consisting of two CGG triplets . Other members of this family, such as Leu3p, also recognize CGG triplets but when oriented in opposite directions, an everted repeat . This implies that the two zinc clusters of Leu3p bound to an everted repeat must be oriented in opposite directions to those of Gal4p or Ppr1p bound to inverted repeats . In order to map the domain responsible for proper orientation of the zinc clusters of Leu3p, we constructed chimeric proteins between Leu3p and Ppr1p and tested their binding to a Leu3p and a Ppr1p site . Our results show that the linker region, which bridges the zinc cluster to the dimerization domain, specifies binding of Leu3p to an everted repeat . We propose that the Leu3p linker projects the two zinc clusters of a Leu3p homodimer in opposite directions allowing binding to everted repeats.

J Biol Chem, 1998 Jul 17, 273(29), 18481 - 9
Cloning and characterization of shk2, a gene encoding a novel p21-activated protein kinase from fission yeast; Yang P et al.; We describe the characterization of a novel gene, shk2, encoding a second p21(cdc42/rac)-activated protein kinase (PAK) homolog in fission yeast . Like other known PAKs, Shk2 binds to Cdc42 in vivo and in vitro . While overexpression of either shk2 or cdc42 alone does not impair growth of wild type fission yeast cells, cooverexpression of the two genes is toxic and leads to highly aberrant cell morphology, providing evidence for functional interaction between Cdc42 and Shk2 proteins in vivo . Fission yeast shk2 null mutants are viable and exhibit no obvious phenotypic defects . Overexpression of shk2 restores viability and normal morphology but not full mating competence to fission yeast cells carrying a shk1 null mutation . Additional genetic data suggest that Shk2, like Cdc42 and Shk1, participates in Ras-dependent morphological control and mating response pathways in fission yeast . We also show that overexpression of byr2, a gene encoding a Ste11/MAPK kinase kinase homolog, suppresses the mating defect of cells partially defective for Shk1 function, providing evidence of a link between PAKs and mitogen-activated protein kinase signaling in fission yeast . Taken together, our results suggest that Shk2 is partially overlapping in function with Shk1, with Shk1 being the dominant protein in function.

J Biol Chem, 1998 Jul 17, 273(29), 18470 - 80
Characterization of a temperature-sensitive yeast vacuolar ATPase mutant with defects in actin distribution and bud morphology; Zhang JW et al.; The 27-kDa E subunit, encoded by the VMA4 gene, is a peripheral membrane subunit of the yeast vacuolar H+-ATPase . We have randomly mutagenized the VMA4 gene in order to examine the structure and function of the 27-kDa subunit . Cells lacking a functional VMA4 gene are unable to grow at pH > 7 or in elevated concentrations of CaCl2 . Plasmid-borne, mutagenized vma4 genes were screened for failure to complement these phenotypes . Mutants producing Vma4 proteins detectable by immunoblot were selected; one (vma4-1(ts)) is temperature conditional, exhibiting the Vma- phenotype only at elevated temperature (37 degreesC) . Sequencing revealed that a single point mutation, D145G, was responsible for the phenotypes of the vma4-1(ts) allele . The unassembled 27-kDa subunit made in the vma4-1(ts) cells is rapidly degraded, particularly at 37 degreesC, but can be protected from degradation by prior assembly into the V-ATPase complex . In purified vacuolar vesicles from the mutant cells, the peripheral subunits are localized to the vacuolar membrane at decreased levels and a comparably decreased level of ATPase activity (14% of the activity in wild-type vesicles) is observed . When vma4-1(ts) mutant cells are shifted to pH 7.5 medium at 37 degrees C, the cells become enlarged and exhibit multiple large buds, elongated buds, and other abnormal morphologies, together with delocalization of actin and chitin, within 4 h . These phenotypes suggest connections between the vacuolar ATPase, bud morphology, and cytokinesis that had not been recognized previously.

J Biol Chem, 1998 Jul 17, 273(29), 18389 - 93
Mt-Hsp70 homolog, Ssc2p, required for maturation of yeast frataxin and mitochondrial iron homeostasis; Knight SA et al.; Here we show that the yeast mitochondrial chaperone Ssc2p, a homolog of mt-Hsp70, plays a critical role in mitochondrial iron homeostasis . Yeast with ssc2-1 mutations were identified by a screen for altered iron-dependent gene regulation and mitochondrial dysfunction . These mutants exhibit increased cellular iron uptake, and the iron accumulates exclusively within mitochondria . Yfh1p is homologous to frataxin, the human protein implicated in the neurodegenerative disease, Friedreich's ataxia . Like mutants of yfh1, ssc2-1 mutants accumulate vast quantities of iron in mitochondria . Furthermore, using import studies with isolated mitochondria, we demonstrate a specific role for Ssc2p in the maturation of Yfh1p within this organelle . This function for a mitochondrial Hsp70 chaperone is likely to be conserved, implying that a human homolog of Ssc2p may be involved in iron homeostasis and in neurodegenerative disease.

J Biol Chem, 1998 Jul 17, 273(29), 18282 - 7
Differential exposure of surface epitopes in the beta-strand region of LOOP1 of the yeast H+-ATPase during catalysis; Seto-Young D et al.; The plasma membrane H+-ATPase of yeast assumes distinct conformational states during its catalytic cycle . To better understand structural changes in the LOOP1 domain, a catalytically important cytoplasmic loop segment linking transmembrane segments 2 and 3, surface epitopes were examined at different stages of catalysis . A polyclonal rabbit antibody was prepared to a fusion protein consisting of LOOP1 and the maltose binding protein . This antibody was affinity-purified to produce a LOOP1-specific fraction that could be used in competition enzyme-linked immunosorbent assays to assess surface exposure of the LOOP1 epitopes . It was found that in an E1 conformation stabilized with either adenosine 5'-(beta,gamma -imino)triphosphate (AMP-PNP) or ADP, less than 10% of the LOOP1 epitopes were accessible on native enzyme . However, when the enzyme was stabilized in an E2-state with ATP plus vanadate, approximately 40% of the surface epitopes on LOOP1 became accessible to antibody . The remaining 60% of the LOOP1 epitopes were fully occluded in the native enzyme and never showed surface exposure . Enzyme-linked immunosorbent assays utilizing fusion proteins consisting of LOOP1 subdomains demonstrated that all of the available epitopes were contained in the beta-strand region (Glu-195-- Val-267) of LOOP1 . The epitopes that were differentially exposed during catalysis were included in regions upstream and downstream of the highly conserved TGES sequence . Our results suggest that during catalysis either the beta-strand region of LOOP1 or an interacting domain undergoes substantial structural rearrangement that facilitates epitope exposure.

Mol Cell, 1998 Jan, 1(2), 223 - 34
Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications; Yang Q et al.; We have calculated a three-dimensional map of the yeast nuclear pore complex (yNPC) from frozen-hydrated specimens, thereby providing a direct comparison with the vertebrate NPC . Overall, the smaller yNPC is comprised of an octagonal inner spoke ring that is anchored within the nuclear envelope by a novel membrane-interacting ring . In addition, a cylindrical transporter is located centrally within the spokes and exhibits a variable radial expansion in projection that may reflect gating . The inner spoke ring, a transmembrane spoke domain, and the transporter are conserved between yeast and vertebrates; hence, they are required to form a functional NPC . However, significant alterations in NPC architecture have arisen during evolution that may be correlated with differences in nuclear transport regulation or mitotic behavior.

Mol Cell, 1998 Jan, 1(2), 161 - 70
The ERO1 gene of yeast is required for oxidation of protein dithiols in the endoplasmic reticulum; Frand AR et al.; We describe a conserved yeast gene, ERO1, that is induced by the unfolded protein response and encodes a novel glycoprotein required for oxidative protein folding in the ER . In a temperature-sensitive ero1-1 mutant, newly synthesized carboxypeptidase Y is retained in the ER and lacks disulfide bonds, as shown by thiol modification with AMS . ERO1 apparently determines cellular oxidizing capacity since mutation of ERO1 causes hypersensitivity to the reductant DTT, whereas overexpression of ERO1 confers resistance to DTT . Moreover, the oxidant diamide can restore growth and secretion in ero1 mutants . Genetic tests distinguish the essential function of ERO1 from that of PDI1 . We show that glutathione is not required for CPY folding and conclude that Ero1p functions in a novel mechanism that sustains the ER oxidizing potential, supporting net formation of protein disulfide bonds.

Mol Pharmacol, 1998 Jul, 54(1), 8 - 13
Yeast expressed cytochrome P450 2D6 (CYP2D6) exposed on the external face of plasma membrane is functionally competent; Loeper J et al.; CYP2D6, a xenobiotic metabolizing cytochrome P450 (P450), was found to be present in significant amount on the outer face of cell plasma membrane in addition to the regular microsomal location . Present work demonstrates that this external P450 is catalytically competent and that activity is supported by NADPH-P450 reductase present on the inner face of plasma membrane . Purified plasma membranes from yeast expressing CYP2D6 sustained NADPH- and cumene hydroperoxide-dependent dextromethorphan demethylation and NADPH-cytochrome c activity confirming previous observations in human hepatocytes . CYP2D6 found on the outside of plasma membrane (by differential immuno-inhibition and acidic shift assays on transformed spheroplasts) was catalytically competent at the cell surface for NADPH-supported activities . Anti-yeast P450-reductase antibodies inhibited neither CYP2D6 nor P450-reductase activities upon incubation with intact spheroplasts . In contrast, both activities were inhibited on isolated plasma membrane fragments . This highly suggested a cytosolic-orientation of the plasma membrane P450-reductase . This finding was confirmed by immunostaining in confocal microscopy . Finally, gene deletion of P450-reductase caused a complete loss of plasma membrane NADPH-supported CYP2D6 activity, which suggests that the reductase participates to some degree in the transmembrane electron transfer chain . This work illustrates that the outside-exposed plasma membrane CYP2D6 is active and may play an important metabolic role.

Mol Biol Cell, 1998 Jul, 9(7), 1833 - 45
Multiple domains of fission yeast Cdc19p (MCM2) are required for its association with the core MCM complex; Sherman DA et al.; The members of the MCM protein family are essential eukaryotic DNA replication factors that form a six-member protein complex . In this study, we use antibodies to four MCM proteins to investigate the structure of and requirements for the formation of fission yeast MCM complexes in vivo, with particular regard to Cdc19p (MCM2) . Gel filtration analysis shows that the MCM protein complexes are unstable and can be broken down to subcomplexes . Using coimmunoprecipitation, we find that Mis5p (MCM6) and Cdc21p (MCM4) are tightly associated with one another in a core complex with which Cdc19p loosely associates . Assembly of Cdc19p with the core depends upon Cdc21p . Interestingly, there is no obvious change in Cdc19p-containing MCM complexes through the cell cycle . Using a panel of Cdc19p mutants, we find that multiple domains of Cdc19p are required for MCM binding . These studies indicate that MCM complexes in fission yeast have distinct substructures, which may be relevant for function.

Mol Biol Cell, 1998 Jul, 9(7), 1757 - 71
Identification of novel temperature-sensitive lethal alleles in essential beta-tubulin and nonessential alpha 2-tubulin genes as fission yeast polarity mutants; Radcliffe P et al.; We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1-15) . Genetic analysis indicates that alp2 is allelic to atb2 (one of two alpha-tubulin genes) and alp12 to nda3 (the single beta-tubulin gene) . atb2(+) is nonessential, and the ts atb2 mutations we have isolated are dominant as expected . We sequenced two alleles of ts atb2 and one allele of ts nda3 . In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between alpha/beta-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule . The ts nda3 mutant is highly sensitive to altered gene dosage of atb2(+); overexpression of atb2(+) lowers the restrictive temperature, and, conversely, deletion rescues ts . Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability . Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

Mol Biol Cell, 1998 Jul, 9(7), 1741 - 56
The yeast dynactin complex is involved in partitioning the mitotic spindle between mother and daughter cells during anaphase B; Kahana JA et al.; Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin . We have cloned and characterized a novel gene, NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150(glued) . Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Delta strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell . Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck . Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase . Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p . Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle . Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

Mol Biol Cell, 1998 Jul, 9(7), 1725 - 39
Dominant negative alleles of SEC10 reveal distinct domains involved in secretion and morphogenesis in yeast; Roth D et al.; The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells . Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeast Saccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion . In this study we have addressed the function of one member of the exocyst complex, Sec10p . We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression . Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p . One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component . Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles . The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect . Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother-daughter connection during cell cycle progression.

J Virol, 1998 Aug, 72(8), 6944 - 9
Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system; Bacharach E et al.; We have used the yeast three-hybrid system (D . J . SenGupta, B . Zhang, B . Kraemer, P . Pochart, S . Fields, and M . Wickens, Proc . Natl . Acad . Sci . USA 93:8496-8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVPsi) . Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae . Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVPsi signal containing four stem-loop structures . Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene . Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag . These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions.

Virology, 1998 Jul 5, 246(2), 266 - 76
Pleiotropic effects of HIV-1 protein R (Vpr) on morphogenesis and cell survival in fission yeast and antagonism by pentoxifylline; Zhao Y et al.; Expression of HIV-1 Vpr causes cell cycle G2 arrest, change in cell shape, and cell death over a large evolutionary distance ranging from human to yeast cells . As a step toward understanding these highly conserved Vpr functions, we have examined the effect of Vpr on cytoskeletal elements and the viability of fission yeast . We demonstrate that the changes in cell morphology induced by Vpr in fission yeast are caused by several underlying cellular abnormalities, including increased biosynthesis of chitin in the cell wall, disruption of the actin cytoskeleton, and altered polarity for cell growth . The extent of these cellular alterations and cell survival correlates with the level of vpr expression . Accompanying cell death, Vpr induces aberrant nuclear morphologies in fission yeast which are similar to those found during the apoptosis induced by Vpr in mammalian cells . The Vpr-induced cytopathic effects and cell death can be suppressed by treatment with pentoxifylline, a compound that inhibits HIV-1 viral replication and suppresses Vpr-induced cell cycle G2 arrest in human and fission yeast cells . The results presented here suggest that pentoxifylline suppresses the effects of Vpr by blocking interactions of Vpr with cellular proteins . Given that pentoxifylline has potential therapeutic value in blocking the effects of Vpr in HIV-infected patients, understanding the molecular mechanisms by which pentoxifylline antagonizes Vpr may have general implications for HIV therapy.

Carcinogenesis, 1998 May, 19(5), 741 - 6
Ultraviolet-light induced p53 mutational spectrum in yeast is indistinguishable from p53 mutations in human skin cancer; Inga A et al.; Ultraviolet (UV) light has been associated with the development of human non-melanoma skin cancers (NMSC) . Such cancers often exhibit mutations in the p53 tumour suppressor gene . In order to determine the UV-induced p53 mutation spectrum, a yeast expression vector that harbours a human wild-type p53 cDNA was UV-irradiated in vitro and transfected into a yeast strain that contained the ADE2 gene regulated by a p53-responsive promoter . Forty-five mutant clones contained 51 mutations . Seven mutations were tandem base pair substitutions, four of which being CC-->TT, hallmark mutations of UV mutagenesis . Eighty percent (41/51) of the mutations were single or non-tandem base pair substitutions, the majority of which (27/41) were C-->T transitions . Ninety-five percent of such mutations occurred at dipyrimidine sites . Through a rigorous statistical test, the UV-induced p53 mutation spectrum appears to differ significantly (P < 0.008) from the one induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea, and to be indistinguishable from the one observed in NMSC (P = 0.4) . These results demonstrate that the assay allows the determination of carcinogen-specific p53 mutation fingerprints and represents a new tool for molecular epidemiology.

Cell, 1998 Jun 12, 93(6), 1067 - 76
An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast; Ciosk R et al.; Cohesion between sister chromatids during G2 and M phases depends on the "cohesin" protein Scc1p (Mcd1p) . Loss of cohesion at the metaphase to anaphase transition is accompanied by Scc1p's dissociation from chromatids, which depends on proteolysis of Pds1p mediated by a ubiquitin protein ligase called the anaphase promoting complex (APC) . We show that destruction of Pds1p is the APC's sole role in triggering Scc1p's dissociation from chromatids and that Pds1p forms a stable complex with a 180 kDa protein called Esp1p, which is essential for the dissociation of Scc1p from sister chromatids and for their separation . We propose that the APC promotes sister separation not by destroying cohesins but instead by liberating the "sister-separating" Esp1 protein from its inhibitor Pds1p.

Cell, 1998 Jun 26, 93(7), 1241 - 52
A critical role for amino-terminal glutamine/asparagine repeats in the formation and propagation of a yeast prion; DePace AH et al.; The yeast {PSI+} factor propagates by a prion-like mechanism involving self-replicating Sup35p amyloids . We identified multiple Sup35p mutants that either are poorly recruited into, or cause curing of, wildtype amyloids in vivo . In vitro, these mutants showed markedly decreased rates of amyloid formation, strongly supporting the protein-only prion hypothesis . Kinetic analysis suggests that the prion state replicates by accelerating slow conformational changes rather than by providing stable nuclei . Strikingly, our mutations map exclusively within a short glutamine/asparagine-rich region of Sup35p, and all but one occur at polar residues . Even after replacement of this region with polyglutamine, Sup35p retains its ability to form amyloids . These and other considerations suggest similarities between the prion-like propagation of {PSI+} and polyglutamine-mediated pathogenesis of several neurodegenerative diseases.

Biol Pharm Bull, 1998 Jun, 21(6), 631 - 3
Effect of over-expressed hsp26 on cell growth of yeast; Unno K et al.; Mammalian small heat shock protein (s-hsp) has been suggested to participate not only in stress tolerance but also in the growth regulation and differentiation of cells . To confirm the role of s-hsp in cell growth, we investigated the relationship between the expression of hsp26 and yeast cell growth . Cells lacking constitutive hsp70, ssa1ssa2, have been known to have a poor growth rate and to over-express hsp26 and some other hsps . We obtained several cell clones of ssa1ssa2 whose doubling times were different from one another . The amount of hsp26 was closely linked to the doubling time of ssa1ssa2 cells . This result suggests that the expression of hsp26 modulates the growth rate of yeast cells lacking constitutive hsp70, similarly to mammalian cells.

Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7945 - 50
Activity of the yeast MNN1 alpha-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases; Wiggins CA et al.; A wide diversity of biological molecules are modified by the addition of sugar residues, and a large number of glycosyltransferases have been identified that are responsible for these reactions . Despite catalyzing closely related reactions, many of these transferases show little apparent sequence homology . By comparing two apparently unrelated families of yeast Golgi mannosyltransferases, a short motif containing two aspartate residues was observed that was conserved in both groups of proteins . Mutagenesis of one of the members of these families, the alpha-1, 3-mannosyltransferase Mnn1p, showed that altering either of these aspartates eliminates all enzymatic activity . These changes do not appear to affect the overall folding and assembly of Mnn1p . A similar aspartate-containing sequence was found to be conserved in a diverse range of other glycosyltransferase families, much more frequently than would be expected by chance, suggesting that it is a feature of the catalytic site, or an element of a structural fold, shared by many glycosyltransferases.

Biophys Chem, 1998 May 5, 72(1-2), 185 - 200
Mathematical model of the fission yeast cell cycle with checkpoint controls at the G1/S, G2/M and metaphase/anaphase transitions; Novak B et al.; All events of the fission yeast cell cycle can be orchestrated by fluctuations of a single cyclin-dependent protein kinase, the Cdc13/Cdc2 heterodimer . The G1/S transition is controlled by interactions of Cdc13/Cdc2 and its stoichiometric inhibitor, Rum1 . The G2/M transition is regulated by a kinase-phosphatase pair, Wee1 and Cdc25, which determine the phosphorylation state of the Tyr-15 residue of Cdc2 . The meta/anaphase transition is controlled by interactions between Cdc13/Cdc2 and the anaphase promoting complex, which labels Cdc13 subunits for proteolysis . We construct a mathematical model of fission yeast growth and division that encompasses all three crucial checkpoint controls . By numerical simulations we show that the model is consistent with a broad selection of cell cycle mutants, and we predict the phenotypes of several multiple-mutant strains that have not yet been constructed.

Biophys Chem, 1998 May 5, 72(1-2), 37 - 47
Traveling waves in yeast extract and in cultures of Dictyostelium discoideum; Muller SC et al.; Biological self-organization was investigated in a biochemical and a cellular system: yeast extract and cultures of the slime mold Dictyostelium discoideum . In both systems traveling reaction-diffusion waves occur in response to oscillatory reactions . Glycolytic degradation of sugar in a yeast extract leads to the spontaneous formation of NADH and proton waves . Manipulation of the adenine nucleotide pool by addition of purified plasma membrane ATPase favors the formation of both reaction-diffusion waves and phase waves . The results indicate that the energy charge has an important impact for the dynamics of glycolytic patterns . When affecting the lower part of glycolysis by pyruvate addition the frequency of wave generation was increased with concomitant formation of rotating NADH and proton spirals . During morphogenesis of the cellular system Dictyostelium discoideum, circular and spiral shaped aggregation patterns of motile amoeboid cells form in response to traveling cAMP waves . Velocity analysis of the cell movements reveals that the cAMP waves guide the cells towards the site of wave initiation along optimized trajectories . The minimization of aggregation paths is based on a mechanism exploiting general properties of excitation waves . The resulting aggregation territories are reminiscent of Voronoi diagrams.

Curr Biol, 1998 Jun 18, 8(13), 787 - 90
Sif2p interacts with Sir4p amino-terminal domain and antagonizes telomeric silencing in yeast; Cockell M et al.; Several regions of the Saccharomyces cerevisiae genome are subject to position-dependent transcriptional repression mediated by a multi-component nucleosome-binding complex of silent information regulator proteins (Sir2p, Sir3p and Sir4p) . These proteins are present in limiting amounts in the nucleus and are targeted to specific chromosomal regions by interaction with sequence-specific DNA-binding factors . Different sites of repression compete for Sir complexes, although it is not known how Sir distribution is regulated . In a screen for factors that interact with Sir4p amino terminus, we have cloned SIF2, which encodes a WD40-repeat-containing factor that disrupts telomeric silencing when overexpressed . In contrast to deletion of SIR4, SIF2 deletion improved telomeric repression, suggesting that under normal conditions Sif2p antagonizes Sir4p function at telomeres . Sif2p overexpression altered the subnuclear localization of Sir4p, but not its protein expression level, suggesting that Sif2p may recruit Sir4p to nontelomeric sites or repression . The sif2 mutant strains were hypersensitive to a range of stress conditions, but did not have decreased viability and did not alter repression in the rDNA . In conclusion, Sif2p resembles the Sir4p regulatory proteins Sir1p and Uth4p in that it competes for the functional assembly of Sir4p at telomeres, yet unlike Sir1p or Uth4p, it does not target Sir4p to either mating-type or rDNA loci.

Mol Cell, 1998 Jun, 1(7), 1051 - 5
A single amino acid change in the yeast retrotransposon Ty5 abolishes targeting to silent chromatin; Gai X et al.; Many retrotransposons and retroviruses are thought to select integration sites through interactions with specific chromosomal proteins . In yeast, the Ty5 retrotransposon integrates preferentially with regions bound by silent chromatin, namely the telomeres and the HMR and HML mating loci . A Ty5 mutant (M3) was identified with an approximately 20-fold decrease in targeted integration as measured by a plasmid-based targeting assay . Often chromosomal insertions generated by M3, none were located at the telomeres or silent mating loci . A single amino acid change at the boundary of integrase and reverse transcriptase is responsible for the mutant phenotype . We predict that this mutation lies within a targeting domain that mediates Ty5 target choice by interacting with a component of silent chromatin.

Mol Cell, 1998 Jun, 1(7), 1021 - 31
SAP30, a novel protein conserved between human and yeast, is a component of a histone deacetylase complex; Zhang Y et al.; Histone acetylation plays a key role in the regulation of eukaryotic gene expression . Recently, histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription . Here, we identify SAP30 as a novel component of the human histone deacetylase complex that includes Sin3, the histone deacetylases HDAC1 and HDAC2, histone binding proteins RbAp46 and RbAp48, as well as other polypeptides . Moreover, we describe a SAP30 homolog in yeast that is functionally related to Sin3 and the histone deacetylase Rpd3 . The human SAP30 complex is active in deacetylating core histone octamers, but inactive in deacetylating nucleosomal histones due to the inability of the histone binding proteins RbAp46 and RbAp48 to gain access to nucleosomal histones . These results define SAP30 as a component of a histone deacetylase complex conserved among eukaryotic organisms.

J Biol Chem, 1998 Jul 10, 273(28), 17859 - 64
The N-terminal region of yeast TFIIB contains two adjacent functional domains involved in stable RNA polymerase II binding and transcription start site selection; Pardee TS et al.; The general transcription factor IIB (TFIIB) is required for accurate and efficient transcription of protein-coding genes by RNA polymerase II (RNAPII) . To define functional domains in the highly conserved N-terminal region of TFIIB, we have analyzed 14 site-directed substitution mutants of yeast TFIIB for their ability to support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and to form stable complexes with purified RNAPII in vitro . Mutations impairing the formation of stable TFIIB.RNAPII complexes mapped to the zinc ribbon fold, whereas mutations conferring downstream shifts in transcription start site selection were identified at multiple positions within a highly conserved homology block adjacent and C-terminal to the zinc ribbon . These results demonstrate that the N-terminal region of yeast TFIIB contains two separable and adjacent functional domains involved in stable RNAPII binding and transcription start site selection, suggesting that downstream shifts in transcription start site selection do not result from impairment of stable TFIIB.RNAPII binding . We discuss models for yeast start site selection in which TFIIB may affect the ability of preinitiation complexes to interact with downstream DNA or to affect start site recognition by a scanning polymerase.

J Biol Chem, 1998 Jul 10, 273(28), 17411 - 7
Structure-function relationships in membrane segment 5 of the yeast Pma1 H+-ATPase; Dutra MB et al.; Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+, K+-ATPase of animal cells . In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis . Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R . K., Rao, R., and Slayman, C . W . (1991) J . Biol . Chem . 266, 7940-7949) . Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals . The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail . One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle . Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate . Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis . The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase . This change was substantially smaller than the 13-30-fold decrease in K+ affinity seen in corresponding mutants of the Na+, K+-ATPase (Arguello, J . M., and Lingrel, J . B (1995) J . Biol . Chem . 270, 22764-22771) . Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle . It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative alpha-helix.

EMBO J, 1998 Jul 1, 17(13), 3738 - 46
Depletion of yeast RNase III blocks correct U2 3' end formation and results in polyadenylated but functional U2 snRNA; Abou Elela S et al.; Yeast U2 snRNA is transcribed by RNA polymerase II to generate a single non-polyadenylated transcript . A temperature-sensitive yeast strain carrying a disruption in RNT1, the gene encoding a homolog of RNase III, produces 3'-extended U2 that is polyadenylated . The U2 3'-flanking region contains a putative stem-loop that is recognized and cleaved at two sites by recombinant GST-Rnt1 protein in vitro . Removal of sequences comprising the stem-loop structure blocks cleavage in vitro and mimics the effects of Rnt1 depletion in vivo . Strains carrying a U2 gene lacking the Rnt1 cleavage site produce only polyadenylated U2 snRNA, and yet are not impaired in growth or splicing . The results suggest that eukaryotic RNase III may be a general factor in snRNA processing, and demonstrate that polyadenylation is not incompatible with snRNA function in yeast.

Heredity, 1998 May, 80 ( Pt 5), 538 - 52
Gene conversion disparity in yeast: its extent, multiple origins, and effects on allele frequencies; Lamb BC; The extent of disparity in gene conversion direction in yeast (Saccharomyces cerevisiae) is important for recombination mechanisms and for effects of conversion on allele frequencies in populations . An analysis of published and unpublished data demonstrates that yeast frequently shows significant and extensive conversion disparity, contrary to many published statements . All types of mutation--base-substitutions, frameshifts and longer deletions and additions--can show significant 6:2/2:6 and/or 5:3/3:5 disparity . There was little correlation between the occurrence of 6:2/2:6 and 5:3/3:5 disparities; when both were significant, they were more often in opposite directions than in the same direction . Surprisingly, there was little correlation between a mutation's molecular nature and its disparity properties, which generally seem unpredictable . Disparity in yeast has multiple origins . From the equations discussed, all disparity types can be explained by one or more of: correction direction disparity, chromatid invasion disparity (including cases caused by different frequencies of double-strand breaks or gaps in nonsister homologous chromatids), strand invasion disparity, and different correction frequencies for the two types of mispair for a heterozygous mutation . Levels of overall disparity and of conversion frequency mean that conversion must often change allele frequencies in sexually reproducing yeast populations.

Genetics, 1998 Jul, 149(3), 1235 - 50
Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells; Galli A et al.; Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events . We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events . Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2 . We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2 . Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively . I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair . In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells . To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage . UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases . Both forms of radiation, however, induced DEL recombination in dividing cells . The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway . Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template . Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage.

Genetics, 1998 Jul, 149(3), 1205 - 19
Distribution of a limited Sir2 protein pool regulates the strength of yeast rDNA silencing and is modulated by Sir4p; Smith JS et al.; Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1 . Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2 . SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing . In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels . The endogenous level of Sir2p was shown to be limiting for rDNA silencing . Furthermore, small changes in Sir2p levels altered rDNA silencing strength . In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2 . Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p . This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2 . Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing . We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein.

EMBO J, 1998 Jul 1, 17(13), 3556 - 64
Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation; Smith A et al.; Yeast cAMP-dependent protein kinase (PKA) activity is essential for growth and antagonizes induction of the general stress response as well as accumulation of glycogen stores . Previous studies have suggested that the PKA effects on the two latter processes result in part from transcription repression . Here we show that transcription derepression that accompanies PKA depletion is dependent upon the presence of two redundant Zn2+-finger transcription factors, Msn2p and Msn4p . The Msn2p and Msn4p proteins were shown previously to act as positive transcriptional factors in the stress response pathway, and our results suggest that Msn2p and Msn4p also mediate PKA-dependent effects on stress response as well as glycogen accumulation genes . Interestingly, PKA activity is dispensable in a strain lacking Msn2p and Msn4p activity . Thus, Msn2p and Msn4p may antagonize PKAdependent growth by stimulating expression of genes that inhibit growth . In agreement with this model, Msn2p/Msn4p function is required for expression of a gene, YAK1, previously shown to antagonize PKA-dependent growth . These results suggest that Msn2p/Msn4p-dependent gene expression may account for all, or at least most, of the pleiotropic effects of yeast PKA, including growth regulation, response to stress and carbohydrate store accumulation.

Anal Biochem, 1998 Jun 15, 260(1), 80 - 4
Temperature gradient chamber for relative growth rate analysis of yeast; Fogel GB et al.; Relative growth is often used as a phenotypic measure to distinguish mutant and wild-type yeast or bacterial strains . Differential growth as a function of temperature is a convenient and accurate means of analyzing differences between strains . Slight differences in the genotypes of two strains frequently result in differential growth of the two strains as a function of temperature . We have developed a chamber for the simultaneous growth of multiple strains in microtiter plates along a temperature gradient . Image analysis was used to determine colony area and number at various times as a function of temperature . This chamber reduces the time required and increases the accuracy in measuring growth as a function of temperature . This occurs by allowing relative growth to be measured along a temperature gradient where all other conditions are constant . Two strains of yeast (Saccharomyces cerevisiae) with a known difference in temperature dependence of growth were used to demonstrate the performance of this chamber.

Anal Biochem, 1998 Jun 15, 260(1), 38 - 43
Affinity purification of yeast cytochrome oxidase with biotinylated subunits 4, 5, or 6; Glerum DM et al.; Null mutants in COX4, COX5a, or COX6, which encode subunits 4, 5, and 6 of yeast cytochrome oxidase are blocked in assembly of the enzyme . The mutants are complemented by gene constructs expressing cytochrome oxidase subunits with a carboxyl terminal extension containing a biotinylation signal sequence . Spectra and enzyme activities of mitochondria from transformants expressing a biotinylated subunit indicate restoration of a functional cytochrome oxidase . Biotinylated cytochrome oxidase can be affinity-purified from mitochondrial extracts by fractionation on a monomeric avidin column . This method can be used to purify the enzyme from small amounts of starting material.

Biosci Biotechnol Biochem, 1998 May, 62(5), 986 - 9
Salicylic acid induces a cytosolic Ca2+ elevation in yeast; Mori IC et al.; Cytosolic free calcium ion concentration ({Ca2+}cyt) after a salicylic acid (SA)-stimulus was monitored in cells of the yeast Saccharomyces cerevisiae expressing apoaequorin, which constitutes a Ca(2+)-sensitive luminescent protein, aequorin, when combined with coelenterazine . SA induced a transient {Ca2+}cyt elevation that was dependent on the concentration of SA and pH of the SA solution . The SA-induced {Ca2+}cyt elevation was not reduced in Ca(2+)-deficient medium, suggesting that Ca2+ was mobilized from an intracellular Ca2+ store(s) . Benzoic acid, butyric acid and sorbic acid did not induced a {Ca2+}cyt elevation.

Genome Res, 1998 Jun, 8(6), 666 - 72
Rescue of targeted regions of mammalian chromosomes by in vivo recombination in yeast; Kouprina N et al.; In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions . In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes . Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element . This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts . Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region . These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment . Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture . Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in approximately 1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system.

Genome Res, 1998 Jun, 8(6), 590 - 8
Large-scale taxonomic profiling of eukaryotic model organisms: a comparison of orthologous proteins encoded by the human, fly, nematode, and yeast genomes; Mushegian AR et al.; Comparisons of DNA and protein sequences between humans and model organisms, including the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, are a significant source of information about the function of human genes and proteins in both normal and disease states . Important questions regarding cross-species sequence comparison remain unanswered, including (1) the fraction of the metabolic, signaling, and regulatory pathways that is shared by humans and the various model organisms; and (2) the validity of functional inferences based on sequence homology . We addressed these questions by analyzing the available fractions of human, fly, nematode, and yeast genomes for orthologous protein-coding genes, applying strict criteria to distinguish between candidate orthologous and paralogous proteins . Forty-two quartets of proteins could be identified as candidate orthologs . Twenty-four Drosophila protein sequences were more similar to their human orthologs than the corresponding nematode proteins . Analysis of sequence substitutions and evolutionary distances in this data set revealed that most C . elegans genes are evolving more rapidly than Drosophila genes, suggesting that unequal evolutionary rates may contribute to the differences in similarity to human protein sequences . The available fraction of Drosophila proteins appears to lack representatives of many protein families and domains, reflecting the relative paucity of genomic data from this species.

Mol Gen Genet, 1998 May, 258(3), 215 - 21
Expression of the yeast BFR2 gene is regulated at the transcriptional level and through degradation of its product; Chabane S et al.; The essential Saccharomyces cerevisiae gene BFR2 has been isolated as a high-copy suppressor of the growth defects induced by Brefeldin A, a drug that disrupts the Golgi apparatus and its protein influx . Furthermore, BFR2 has been found to display genetic interactions with four mutations affecting protein transport to the Golgi apparatus . Here we show that the level of BFR2 mRNA rapidly increased over fivefold in response to cold shock, and over threefold following nutrient replenishment by dilution of cells from exhausted to fresh minimal medium . During subsequent growth, the transcript level returned to its basal values, except for a transient drop toward the end of the exponential phase . The early burst of transcription was not caused by toxic compounds in the fresh medium, or by synchrony among cells that had simultaneously entered their first cell cycle . The BFR2 gene product (Bfr2p) was synthesized following the early burst of mRNA, and was no longer produced when the mRNA was back to basal level . Bfr2p was finally degraded after growth became limited, and reached undetectable levels in exhausted medium . Under steady-state conditions of lengthened exponential phase, the intracellular level of Bfr2p remained constant . This peculiar pattern of gene expression suggests that Bfr2p is essential for mass growth or cell proliferation, whereas it is either toxic or not required during nutrient-limited growth.

Mol Gen Genet, 1998 May, 258(3), 183 - 98
Cyclin-specific START events and the G1-phase specificity of arrest by mating factor in budding yeast; Oehlen LJ et al.; The START cell cycle transition in the budding yeast Saccharomyces cerevisiae is catalyzed by the Cdc28 cyclin-dependent kinase associated with Cln-type cyclins . Since ectopic expression of the B-type cyclin CLB5 can efficiently rescue the inviability that results from CLN depletion, we tested the specificity of the CLN and CLB classes of cyclins for promoting START-associated events . Several aspects of the regulation of the mating factor response were compared for cells in which START activity was provided by either Cln-cyclins or Clb5 . Unlike Cln1 and Cln2, high level expression of Clb5 was unable to repress the activity of the mating factor response pathway at START . Downregulation of Far1 protein at START is normal in cln- GAL1::CLB5 cells . Even though the Clb5-Cdc28 kinase activity in cln- GAL1::CLB5 cells is not downregulated in response to mating factor, cells arrest in the first cycle after addition of mating factor with a similar sensitivity as wild-type cells . However, whereas wild-type cells treated with mating factor arrest specifically in G1 phase as unbudded cells with unreplicated DNA (pre-START), most cln- GAL1::CLB5 cells arrest as budded post-START cells with replicated DNA . Our findings demonstrate the ability of post-START cells to arrest in response to mating factor and provide novel evidence for mechanisms that contribute to restrict mating factor-induced arrest in wild-type cells to the G1 phase of the cell cycle.

Genome, 1998 Apr, 41(2), 236 - 43
The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking; Warner TS et al.; Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes . In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions . Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking . We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA . The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes . Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

J Biol Chem, 1998 Jul 3, 273(27), 17012 - 7
Tyrosine and tryptophan act through the same binding site at the dimer interface of yeast chorismate mutase; Schnappauf G et al.; Tyrosine and tryptophan are the regulators of the dimeric yeast chorismate mutase . Biochemical studies reveal two binding sites per molecule for both effectors, tyrosine or tryptophan . A single binding site is built up by helix 8 and helices 4 and 5 of two different subunits . The binding sites have been analyzed in the active enzyme by site directed mutagenesis of critical codons of the coding gene, ARO7 . Gly-141 and Ser-142, which both reside on helix 8, are involved in the binding of tyrosine or tryptophan presumably by interacting specifically with the amino- and carboxylate-groups of these amino acid effectors . Interaction with Thr-145 of helix 8 is required for a strong tyrosine binding to the allosteric site . Replacement of Arg-75, which connects helices 4 and 5 or of Arg-76, which is part of helix 5 by alanine residues, resulted in unregulated enzymes . These two residues are bonded to the carboxylate group and phenolic hydroxyl group of tyrosine, respectively, but do not interact with tryptophan by hydrogen bonding in the crystal structures . Phenylalanine, which has low binding affinity slightly activated the chorismate mutase . A T145V mutant chorismate mutase, however, showed increased activation by phenylalanine . Our results support a mechanism by which tyrosine contracts the allosteric site by interacting with its phenolic hydroxyl group . Tryptophan works in an inverse way by opening the allosteric site through the steric size of its side chain.

J Biol Chem, 1998 Jul 3, 273(27), 16635 - 8
A role for phospholipase D (Pld1p) in growth, secretion, and regulation of membrane lipid synthesis in yeast; Sreenivas A et al.; The SEC14 gene encodes a phosphatidylinositol/phosphatidylcholine transfer protein essential for secretion and growth in yeast (1) . Mutations (cki1, cct1, and cpt1) in the CDP-choline pathway for phosphatidylcholine synthesis suppress the sec14 growth defect (2), permitting sec14(ts) cki1, sec14(ts) cct1, and sec14(ts) cpt1 strains to grow at the sec14(ts) restrictive temperature . Previously, we reported that these double mutant strains also excrete the phospholipid metabolites, choline and inositol (3) . We now report that these choline and inositol excretion phenotypes are eliminated when the SPO14 (PLD1) gene encoding phospholipase D1 is deleted . In contrast to sec14(ts) cki1 strains, sec14(ts) cki1 pld1 strains are not viable at the sec14(ts) restrictive temperature and exhibit a pattern of invertase secretion comparable with sec14(ts) strains . Thus, the PLD1 gene product appears to play an essential role in the suppression of the sec14(ts) defect by CDP-choline pathway mutations, indicating a role for phospholipase D1 in growth and secretion . Furthermore, sec14(ts) strains exhibit elevated Ca2+-independent, phophatidylinositol 4,5-bisphosphate-stimulated phospholipase D activity . We also propose that phospholipase D1-mediated phosphatidylcholine turnover generates a signal that activates transcription of INO1, the structural gene for inositol 1-phosphate synthase.

J Mol Biol, 1998 Jun 19, 279(4), 929 - 43
A disorder-to-order transition coupled to DNA binding in the essential zinc-finger DNA-binding domain of yeast ADR1; Hyre DE et al.; The motional dynamics and solvent-exchange behavior of free and DNA-bound forms of the minimal zinc-finger DNA-binding domain of the yeast transcription factor ADR1 (ADR1-DBD) are investigated using NMR . The parameters measured include the 1H-15N heteronuclear NOE, 15N and 1H T1 relaxation rates, 15N T2 relaxation rates, and solvent-exchange rates . The spin relaxation parameters, spectral density maps, and solvent-exchange behavior show that, exclusive of the N and C termini, three distinct regions of free ADR1-DBD exhibit different motions on multiple timescales . The N-terminal proximal, or accessory, region appears to be unstructured and highly flexible: it exhibits large amplitude motions on a picosecond timescale, little or no protection from solvent exchange, and random-coil proton chemical shifts . The two zinc fingers tumble anisotropically as folded domains, with the tumbling of the individual fingers being only partly correlated to each other, and are modestly protected from solvent exchange except near the tips of the fingers and in the linker joining them . Free ADR1-DBD exhibits exchange broadening around P97 in the proximal region, at the tip of finger 1, and throughout finger 2 . Upon binding, most of the proximal region and both zinc fingers tumble as a single domain and exhibit significantly reduced picosecond timescale motions . This region becomes more protected from solvent exchange . The bound portion of the proximal region is proposed to lie exposed on the surface of the DNA . Exchange broadening remains around P97 but also becomes evident for residues in direct contact with the DNA and in the linker . We conclude that the region of ADR1-DBD essential for high-affinity binding undergoes a disorder-to-order transition upon binding to its cognate DNA and, together with the zinc fingers, forms a cohesive molecular complex with the nucleic acid .

Mutat Res, 1998 Jun 5, 401(1-2), 11 - 25
Utilization of microhomologous recombination in yeast to generate targeting constructs for mammalian genes; Khrebtukova I et al.; We have developed a new procedure utilizing microhomologous recombination in yeast to generate targeting constructs for producing targeted mutations in mice . This procedure is rapid and efficient, and should be directly applicable to all mammalian genes . Moreover, only minimal information about the locus being targeted is required . The feasibility of this approach was demonstrated by producing another allele of the mouse Tg737 polycystic kidney gene .

Biochem J, 1998 Jun 1, 332 ( Pt 2), 583 - 9
Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis; Tijet N et al.; The chemical tagging of a cytochrome P-450-dependent lauric acid omega-hydroxylase from clofibrate-treated Vicia sativa seedlings with {1-14C}11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1 . We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana . The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1 . The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1 . Both E/Z and Z/E configurations of 9, 12-octadecadienoic acids were omega-hydroxylated . Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1) . The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized . Similar to animal omega-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment . Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug . The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.

Yeast, 1998 May, 14(7), 655 - 64
Genomic disruption of six budding yeast genes gives one drastic example of phenotype strain-dependence; Bilsland E et al.; Using PCR to construct disruption cassettes, null alleles of six genes have been created in Saccharomyces cerevisiae . In a FY1679 background, no defects were detected in any of the haploid deletion mutants with respect to growth, gross morphology, or mating . A diploid FY1679-derived delta ygl194c/delta ygl194c homozygous disruptant displayed reduced sporulation . In contrast to the lack of phenotypic consequences of delta yol100w disruptions in the FY1679 background, in the CEN.PK2 strain even a heterozygous disruption of the same gene caused striking effects, very slow vegetative growth and highly impaired sporulation . Tetrad analysis showed YOL100w to be an essential gene in this strain . A copy of the YGL194c or the YOL100w wild-type gene borne on a centromeric episomal plasmid was introduced into a corresponding disruption mutant strain, and in both cases was found to partially complement the defects.

Glycobiology, 1998 Aug, 8(8), 761 - 70
GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid; Canivenc-Gansel E et al.; Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins . Previously, we isolated a GPI deficient yeast mutant accumulating a GPI intermediate containing only two mannoses, suggesting that it has difficulty in adding the third, alpha1,2-linked Man of GPI anchors . The mutant thus displays a similar phenotype as the mammalian mutant cell line S1A-b having a mutation in the PIG-B gene . The yeast mutant, herein named gpi10-1 , contains a mutation in YGL142C, a yeast homolog of the human PIG-B . YGL142C predicts a highly hydrophobic integral membrane protein which by sequence is related to ALG9, a yeast gene required for adding Man in alpha1,2 linkage to N-glycans . Whereas gpi10-1 cells grow at a normal rate and make normal amounts of GPI proteins, the microsomes of gpi10-1 are completely unable to add the third Man in an in vitro assay . Further analysis of the GPI intermediate accumulating in gpi10 shows it to have the structure Manalpha1-6(EtN-P-)Manalpha1-4GlcNalpha1-6(acyl) Inositol-P-lipid . The presence of EtN-P on the alpha1,4-linked Man of GPI anchors is typical of mammalian and a few other organisms but had not been observed in yeast GPI proteins . This additional EtN-P is not only found in the abnormal GPI intermediate of gpi10-1 but is equally present on the complete GPI precursor lipid of wild type cells . Thus, GPI biosynthesis in yeast and mammals proceeds similarly and differs from the pathway described for Trypanosoma brucei in several aspects.

Mol Cell Biol, 1998 Jul, 18(7), 4400 - 6
Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA; Hays SL et al.; The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication . A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52 . We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway . Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA . The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52 . Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U . H . Mortenson, C . Beudixen, I . Sunjeuaric, and R . Rothstein, Proc . Natl . Acad . Sci . USA 93:10729-10734, 1996) . The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins . Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51 . The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1 . Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2 . Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented.

Mol Cell Biol, 1998 Jul, 18(7), 4157 - 64
The C-terminal domain of Sin1 interacts with the SWI-SNF complex in yeast; Perez-Martin J et al.; In the yeast Saccharomyces cerevisiae, the SWI-SNF complex has been proposed to antagonize the repressive effects of chromatin by disrupting nucleosomes . The SIN genes were identified as suppressors of defects in the SWI-SNF complex, and the SIN1 gene encodes an HMG1-like protein that has been proposed to be a component of chromatin . Specific mutations (sin mutations) in both histone H3 and H4 genes produce the same phenotypic effects as do mutations in the SIN1 gene . In this study, we demonstrate that Sin1 and the H3 and H4 histones interact genetically and that the C terminus of Sin1 physically associates with components of the SWI-SNF complex . In addition, we demonstrate that this interaction is blocked in the full-length Sin1 protein by the N-terminal half of the protein . Based on these and additional results, we propose that Sin1 acts as a regulatable bridge between the SWI-SNF complex and the nucleosome.

Mol Cell Biol, 1998 Jul, 18(7), 4053 - 69
Spa2p interacts with cell polarity proteins and signaling components involved in yeast cell morphogenesis; Sheu YJ et al.; The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth . To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p . Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Delta, pea2Delta, and bud6Delta mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes . Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo . Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex . Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton . Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites . One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II . Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function . By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth . Spa2p interacts with Ste11p (MAPK kinase {MEK} kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain . Bud6p interacts with Ste11p . The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling . spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity . We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.

Mol Cell Biol, 1998 Jul, 18(7), 3819 - 28
Molecular mechanism governing heme signaling in yeast: a higher-order complex mediates heme regulation of the transcriptional activator HAP1; Zhang L et al.; Apart from serving as a prosthetic group in globins and enzymes, heme is a key regulator controlling a wide range of molecular and cellular processes involved in oxygen sensing and utilization . To gain insights into molecular mechanisms of heme signaling and oxygen sensing in eukaryotes, we investigated the yeast heme-responsive transcriptional activator HAP1 . HAP1 activity is regulated precisely and tightly by heme . Here we show that in the absence of heme, HAP1 forms a biochemically distinctive higher-order complex . Our data suggest that this complex contains HAP1 and four other cellular proteins including Hsp82 and Ydj1 . The formation of this complex is directly correlated with HAP1 repression in the absence of heme, and mutational or heme disruption of the complex correlates with HAP1 activation, suggesting that this complex is responsible for heme regulation of HAP1 activity . Further, we determined HAP1 domains required for heme regulation: three domains-the dimerization domain, the heme domain, and the HRM7 (heme-responsive motif 7) domain-cooperate to form the higher-order complex and mediate heme regulation . Strikingly, we uncovered a novel function for the HAP1 dimerization domain: it not only allows dimerization but also provides critical functions in heme regulation and transcriptional activation . Our studies provide significant insights into the molecular events leading to heme activation of HAP1 and may shed light on molecular mechanisms of various heme-controlled biological processes in diverse organisms.

Mol Cell Biol, 1998 Jul, 18(7), 3727 - 34
SBA1 encodes a yeast hsp90 cochaperone that is homologous to vertebrate p23 proteins; Fang Y et al.; The Saccharomyces cerevisiae SBA1 gene was cloned by PCR amplification from yeast genomic DNA following its identification as encoding an ortholog of human p23, an Hsp90 cochaperone . The SBA1 gene product is constitutively expressed and nonessential, although a disruption mutant grew more slowly than the wild type at both 18 and 37 degreesC . A double deletion of SBA1 and STI1, encoding an Hsp90 cochaperone, displayed synthetic growth defects . Affinity isolation of histidine-tagged Sba1p (Sba1(His6)) after expression in yeast led to coisolation of Hsp90 and the cyclophilin homolog Cpr6 . Using an in vitro assembly assay, purified Sba1(His6) bound to Hsp90 only in the presence of adenosine 5'-O-(3-thiotriphosphate) or adenyl-imidodiphosphate . Furthermore, interaction between purified Sba1(His6) and Hsp90 in yeast extracts was inhibited by the benzoquinoid ansamycins geldanamycin and macbecin . The in vitro assay was also used to identify residues in Hsp90 that are important for complex formation with Sba1(His6), and residues in both the N-terminal nucleotide binding domain and C-terminal half were characterized . In vivo analysis of known Hsp90 substrate proteins revealed that Sba1 loss of function had only a mild effect on the activity of the tyrosine kinase v-Src and steroid hormone receptors.

J Cell Biol, 1998 Jun 15, 141(6), 1371 - 81
Interaction between mitochondria and the actin cytoskeleton in budding yeast requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p; Boldogh I et al.; Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny . The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud . Sedimentation assays reveal two different components required for mitochondria-actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle . mABP activity is abolished by treatment of mitochondria with high salt . Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity . mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain . Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M . Delannoy, and R.E . Jensen . 1994 . J.Cell Biol . 126:1375-1391) and Mdm10p, (Sogo, L.F., and M.P . Yaffe . 1994 . J.Cell Biol . 126:1361- 1373) are required for these actin-mitochondria interactions . Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity . Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Delta mutants appears to be actin independent . Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement . Mitochondrial motility in mmm1-1 and mdm10Delta mutants is indistinguishable from that in latrunculin-A-treated wild-type cells . We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.

J Cell Biol, 1998 Jun 15, 141(6), 1357 - 70
The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization; Anderson BL et al.; The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions . Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes . Expression of myc-tagged Myo5p in myo3Delta myo5Delta cells fully restores wild-type characteristics . Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth . We find that latrunculin-A-induced depolymerization of F-actin results in loss of Myo5p patches . Moreover, incubation of yeast cells at 37 degrees C results in transient depolarization of both Myo5p patches and the actin cytoskeleton . Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Delta myo5Delta cells and the resulting strains were analyzed for Myo5p function . Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function . In contrast, myo3Delta myo5Delta cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction . These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast . The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p . Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining . Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth . Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant . Our studies support a multistep model for Myo5p targeting in yeast . The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.

FEBS Lett, 1998 May 15, 427(3), 345 - 8
Biological dynamics and distribution of 3-hydroxy fatty acids in the yeast Dipodascopsis uninucleata as investigated by immunofluorescence microscopy . Evidence for a putative regulatory role in the sexual reproductive cycle; Kock JL et al.; Dipodascopsis uninucleata has been recently shown to produce 3-hydroxy polyenoic fatty acids from several exogenous polyenoic fatty acids . In order to examine whether endogenous 3-hydroxy fatty acids (3-OH-FA) may be implicated in the developmental biology of this yeast, we mapped by immunofluorescence microscopy their occurrence in fixed cells with or without cell walls using an antibody raised against 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (3R-HETE), the biotransformation product from arachidonic acid (AA) . This antibody turned out to cross-react with other 3-OH-FA . 3-OH-FA were detected in situ in gametangia, asci, as well as between released ascospores, and proved to be associated with the sexual reproductive stage of the life cycle of the yeast . Acetylsalicylic acid (1 mM), which is known to suppress the formation of 3-OH-FA from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as the sexual phase of the life cycle suggesting a prominent regulatory role of 3-OH-FA for the latter.

Virology, 1998 Jun 5, 245(2), 323 - 30
HHR23A, the human homologue of the yeast repair protein RAD23, interacts specifically with Vpr protein and prevents cell cycle arrest but not the transcriptional effects of Vpr; Gragerov A et al.; Yeast two-hybrid selection of proteins interacting with human immunodeficiency virus type 1 Vpr identified HHR23A, a human homologue of the yeast DNA repair protein RAD23, as a specific interactor . A small 57-amino-acid C-terminal portion of HHR23A was sufficient for Vpr interaction . When introduced into human cells by transfection, full-length HHR23A or its C-terminal fragments were able to alleviate Vpr-induced cell cycle arrest, suggesting that HHR23A may participate in the pathway leading to G2 arrest by Vpr . We have also examined the effects of HHR23 on the recently identified transcription coactivator function of Vpr . The two Vpr functions are independent, since we have identified mutants lacking either the cell cycle arrest or the coactivator function . Our analysis showed that excess of HHR23A does not affect the coactivator function of Vpr, while it affects the cell cycle arresting function . Therefore, a simple sequestering model for Vpr in the presence of excess HHR23A is not supported . We propose that the interaction of HHR23A with Vpr may affect specifically pathways leading to cell cycle regulation.

Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7526 - 31
Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle; Verde F et al.; The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown . We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression . The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family . A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34(cdc2) mitotic kinase . Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis . orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway . Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

Chem Res Toxicol, 1998 Jun, 11(6), 622 - 9
Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay; Breinholt V et al.; A newly developed recombinant yeast strain, in which the human estrogen receptor has been stably integrated into the genome of the yeast, was used to gain information on the estrogenic activity of a large series of dietary flavonoids . Among 23 flavonoids investigated, 8 were found to markedly stimulate the transcriptional activity of the human estrogen receptor in the yeast assay increasing transcriptional activity 5-13-fold above background level, corresponding to EC50 values between 0.1 and 25 microM . Five compounds increased the transcriptional activity 2-5-fold over the control, with EC50 values ranging from 84 to 102 microM, whereas the remaining flavonoids were devoid of activity . The most potent flavonoid estrogens tested were naringenin, apigenin, kaempferol, phloretin, and the four isoflavonoids equol, genistein, daidzein, and biochanin A . With the exception of biochanin A, the main feature required to confer estrogenicity was the presence of a single hydroxyl group in the 4'-position of the B-ring of the flavan nucleus, corresponding to the 4-position on phloretin . The estrogenic potency of the flavonoids was found to be 4 000-4 000 000 times lower than that observed for 17beta-estradiol, when compared on the basis of EC50 values . The estrogenic activity of the dietary flavonoids was further investigated in estrogen-dependent human MCF7 breast cancer cells . In this system several of the flavonoids were likewise capable of mimicking natural estrogens and thereby induce cell proliferation . Similar structural requirements for estrogenic activity were found for the two assays . The present results provide evidence that several of the flavo-estrogens possess estrogenic properties comparable in activity to the well-established isoflavonoid estrogens . The use of Alamar Blue, a vital dye which is metabolically reduced by cellular enzymes to a fluorescent product, was found to greatly simplify the MCF7 cell-based estrogen screen, making this mammalian assay applicable as a large-scale screening tool for estrogenic compounds.

Curr Biol, 1998 May 21, 8(11), 653 - 6
Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres; Laroche T et al.; The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination {1,2} . Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background {3,4} . In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically {5,6} . We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery {7}, but the elements that mediate this localization remained unknown . We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus . These are the first mutants shown to affect the subnuclear localization of telomeres . Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci . In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm . Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

Nat Biotechnol, 1996 Nov, 14(11), 1587 - 91
Inactivation of MET10 in brewer's yeast specifically increases SO2 formation during beer production; Hansen J et al.; Sulfite is widely used as an antioxidant in food production . In beer brewing, sulfite has the additional role of stabilizing the flavor by forming adducts with aldehydes . Inadequate amounts of sulfite are sometimes produced by brewer's yeasts, so means of controlling the sulfite production are desired . In Saccharomyces yeasts, MET10 encodes a subunit of sulfite reductase . Partial or full elimination of MET10 gene activity in a brewer's yeast resulted in increased sulfite accumulation . Beer produced with such yeasts was quite satisfactory and showed increased flavor stability.

J Biol Chem, 1998 Jun 19, 273(25), 15779 - 86
Phosphatidylinositol-4-phosphate 5-kinase localized on the plasma membrane is essential for yeast cell morphogenesis; Homma K et al.; Phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2), an important element in eukaryotic signal transduction, is synthesized either by phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P 5K) from phosphatidylinositol 4-phosphate (PtdIns(4)P) or by phosphatidylinositol-5-phosphate 4-kinase (PtdIns(5)P 4K) from phosphatidylinositol 5-phosphate (PtdIns(5)P) . Two Saccharomyces cerevisiae genes, MSS4 and FAB1, are homologous to mammalian PtdIns(4)P 5Ks and PtdIns(5)P 4Ks . We show here that MSS4 is a functional homolog of mammalian PtdIns(4)P 5K but not of PtdIns(5)P 4K in vivo . We constructed a hemagglutinin epitope-tagged form of Mss4p and found that Mss4p has PtdIns(4)P 5K activity . Immunofluorescent and fractionation studies of the epitope-tagged Mss4p suggest that Mss4p is localized on the plasma membrane, whereas Fab1p is reportedly localized on the vacuolar membrane . A temperature-sensitive mss4-1 mutant was isolated, and its phenotypes at restrictive temperatures were found to include increased cell size, round shape, random distribution of actin patches, and delocalized staining of cell wall chitin . Thus, biochemical and genetic analyses on Mss4p indicated that yeast PtdIns(4)P 5K localized on the plasma membrane is required for actin organization.

J Biol Chem, 1998 Jun 26, 273(26), 16453 - 63
Nop5p is a small nucleolar ribonucleoprotein component required for pre-18 S rRNA processing in yeast; Wu P et al.; We have identified a novel nucleolar protein, Nop5p, that is essential for growth in Saccharomyces cerevisiae . Monoclonal antibodies B47 and 37C12 recognize Nop5p, which has a predicted size of 57 kDa and possesses a KKX repeat motif at its carboxyl terminus . Truncations that removed the KKX motif were functional and localized to the nucleolus, but conferred slow growth at 37 degreesC . Nop5p shows significant sequence homology with yeast Sik1p/Nop56p, and putative homologues in archaebacteria, plants, and human . Depletion of Nop5p in a GAL-NOP5 strain lengthened the doubling time about 5-fold, and selectively reduced steady-state levels of 40 S ribosomal subunits and 18 S rRNA relative to levels of free 60 S subunits and 25 S rRNA . Northern blotting and primer extension analyses showed that Nop5p depletion impairs processing of 35 S pre-rRNA at the A0 and A2 cleavage sites . Nop5p is associated with the small nucleolar RNAs U3, snR13, U14, and U18 . Depletion of Nop5p caused the nucleolar protein Nop1p (yeast fibrillarin) to be localized to the nucleus and cytosol . Also, 37C12 co-immunoprecipitated Nop1p . These results suggest that Nop5p functions with Nop1p in the execution of early pre-rRNA processing steps that lead to formation of 18 S rRNA.

Mol Cell Biol, 1998 Jun, 18(6), 3191 - 200
A chimeric subunit of yeast transcription factor IIIC forms a subcomplex with tau95; Manaud N et al.; The multisubunit yeast transcription factor IIIC (TFIIIC) is a multifunctional protein required for promoter recognition, transcription factor IIIB recruitment, and chromatin antirepression . We report the isolation and characterization of TFC7, an essential gene encoding the 55-kDa polypeptide, tau55, present in affinity-purified TFIIIC . tau55 is a chimeric protein generated by an ancient chromosomal rearrangement . Its C-terminal half is essential for cell viability and sufficient to ensure TFIIIC function in DNA binding and transcription assays . The N-terminal half is nonessential and highly similar to a putative yeast protein encoded on another chromosome and to a cyanobacterial protein of unknown function . Partial deletions of the N-terminal domain impaired tau55 function at a high temperature or in media containing glycerol or ethanol, suggesting a link between PolIII transcription and metabolic pathways . Interestingly, tau55 was found, together with TFIIIC subunit tau95, in a protein complex which was distinct from TFIIIC and which may play a role in the regulation of PolIII transcription, possibly in relation to cell metabolism.

J Biol Chem, 1998 May 15, 273(20), 12612 - 22
ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p; Decottignies A et al.; The Saccharomyces cerevisiae genome encodes 15 full-size ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance) . We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were up-regulated by the PDR1-3 mutation . These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p . The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins . Overexpressed Yor1p was photolabeled by {gamma-32P}2', 3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K0.5 = 45 microM) and inhibited by ATP (KD = 0.3 mM) in plasma membranes . Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (approximately 100 nmol of Pi.min-1.mg-1) . This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 microM) and oligomycin (I50 = 8.5 microg/ml) . In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin . We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B . Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-{6-(NBD)aminocaproyl}phosphatidylethanolamine . Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane . Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.

J Biol Chem, 1998 May 15, 273(20), 12599 - 605
HAT1 and HAT2 proteins are components of a yeast nuclear histone acetyltransferase enzyme specific for free histone H4; Ruiz-Garcia AB et al.; We have analyzed the histone acetyltransferase enzymes obtained from a series of yeast hat1, hat2, and gcn5 single mutants and hat1,hat2 and hat1,gcn5 double mutants . Extracts prepared from both hat1 and hat2 mutant strains specifically lack the following two histone acetyltransferase activities: the well known cytoplasmic type B enzyme and a free histone H4-specific histone acetyltransferase located in the nucleus . The catalytic subunits of both cytoplasmic and nuclear enzymes have identical molecular masses (42 kDa), the same as that of HAT1 . However, the cytoplasmic complex has a molecular mass (150 kDa) greater than that of the nuclear complex (110 kDa) . The possible functions of HAT1 and HAT2 in the yeast nucleus are discussed . In addition, we have detected a yeast histone acetyltransferase not previously described, designated HAT-A4 . This enzyme is located in the nucleus and is able to acetylate free and nucleosome-bound histones H3 and H4 . Finally, we show that the hat1, gcn5 double mutant is viable and does not exhibit a new phenotype, thus suggesting the existence of several histone acetyltransferases with overlapping functions.

J Biol Chem, 1998 May 15, 273(20), 12567 - 75
Jak2-Stat5 interactions analyzed in yeast; Barahmand-Pour F et al.; Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) for nuclear signaling . Here, we have established yeast strains in which an autoactivated Jak2 kinase induces tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding of a concomitantly expressed Stat5 protein . Transcriptional activity of Stat5 on a stably integrated, Stat-dependent reporter gene required the C-terminal fusion of the VP16 transactivation domain . In such yeast strains, the interaction between Jak2 and Stat5 was analyzed without interference by other mammalian proteins involved in regulating Jak-Stat signaling, and mutant versions of both proteins were analyzed for their ability to productively interact . Complexes between Jak2 and Stat5 were found to be stable under stringent co-immunoprecipitation conditions . Deletion of the Jak homology regions 2-7 (JH2-JH7) of Jak2, leaving only the kinase domain (JH1) intact, reduced the ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain caused an increased enzymatic activity . A site-directed R618K mutation in the Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C terminus led to Stat5 hyperphosphorylation . A single phosphotyrosine-SH2 domain interaction was sufficient for the dimerization of Stat5, but such dimers bound to DNA very inefficiently . Together, our data show that yeast cells are appropriate tools for studying Jak-Stat or Stat-Stat interactions . Our mutational analysis suggests that the Stat5 SH2 domain is essential for the interaction with Jak2 and that the kinase domain of Jak2 is sufficient for Jak2-Stat5 interaction . Therefore, the Jak kinase domain may be all that is needed to cause Stat phosphorylation in situations where receptor docking is dispensable.

J Biol Chem, 1998 May 15, 273(20), 12422 - 6
A yeast recombinant aquaporin mutant that is not expressed or mistargeted in Xenopus oocyte can be functionally analyzed in reconstituted proteoliposomes; Lagree V et al.; We have recently identified AQPcic (for aquaporin cicadella), an insect aquaporin found in the digestive tract of homopteran insects and involved in the elimination of water ingested in excess with the dietary sap (Le Caherec, F., Deschamps, S., Delamarche, C., Pellerin, I., Bonnec, G., Guillam, M . T., Gouranton, J., Thomas, D., and Hubert, J . F . (1996) Eur . J . Biochem . 241, 707-715) . Like many other aquaporins, AQPcic is inhibited by mercury reagents . In this study, we have demonstrated that residue Cys82 is essential for mercury inhibition . Another mutant version of AQPcic (AQP-C134S), expression of which in Xenopus laevis failed to produce an active molecule, was successfully expressed in Saccharomyces cerevisiae . Using stopped-flow analysis of reconstituted proteoliposomes, we demonstrated that the biological activity and Hg sensitivity of yeast-expressed wild type and mutant type AQPcic was readily assessed . Therefore, we propose that the yeast system is a valid alternative to Xenopus oocytes for studying particular mutants of aquaporin.

EMBO J, 1998 May 1, 17(9), 2687 - 98
RAD9 and RAD24 define two additive, interacting branches of the DNA damage checkpoint pathway in budding yeast normally required for Rad53 modification and activation; de la Torre-Ruiz MA et al.; In budding yeast, RAD9 and RAD24/RAD17/MEC3 are believed to function upstream of MEC1 and RAD53 in signalling the presence of DNA damage . Deletion of any one of these genes reduces the normal G1/S and G2/M checkpoint delays after UV irradiation, whereas in rad9Delta-rad24Delta cells the G1/S checkpoint is undetectable, although there is a residual G2/M checkpoint . We have shown previously that RAD9 also controls the transcriptional induction of a DNA damage regulon (DDR) . We now report that efficient DDR induction requires all the above-mentioned checkpoint genes . Residual induction of the DDR after UV irradiation observed in all single mutants is not detectable in rad9Delta-rad24Delta . We have examined the G2/M checkpoint and UV sensitivity of single mutants after overexpression of the checkpoint proteins . This analysis indicates that RAD9 and the RAD24 epistasis group can be placed onto two separate, additive branches that converge on MEC1 and RAD53 . Furthermore, MEC3 appears to function downstream of RAD24/RAD17 . The transcriptional response to DNA damage revealed unexpected and specific antagonism between RAD9 and RAD24 . Further support for genetic interaction between RAD9 and RAD24 comes from study of the modification and activation of Rad53 after damage . Evidence for bypass of RAD53 function under some conditions is also presented.

EMBO J, 1998 May 1, 17(9), 2663 - 76
Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export; Snay-Hodge CA et al.; To identify Saccharomyces cerevisiae genes important for nucleocytoplasmic export of messenger RNA, we screened mutant strains to identify those in which poly(A)+ RNA accumulated in nuclei under nonpermissive conditions . We describe the identification of DBP5 as the gene defective in the strain carrying the rat8-1 allele (RAT = ribonucleic acid trafficking) . Dbp5p/Rat8p, a previously uncharacterized member of the DEAD-box family of proteins, is closely related to eukaryotic initiation factor 4A(eIF4A) an RNA helicase essential for protein synthesis initiation . Analysis of protein databases suggests most eukaryotic genomes encode a DEAD-box protein that is probably a homolog of yeast Dbp5p/Rat8p . Temperature-sensitive alleles of DBP5/RAT8 were prepared . In rat8 mutant strains, cells displayed rapid, synchronous accumulation of poly(A)+ RNA in nuclei when shifted to the non-permissive temperature . Dbp5p/Rat8p is located within the cytoplasm and concentrated in the perinuclear region . Analysis of the distribution of Dbp5p/Rat8p in yeast strains where nuclear pore complexes are tightly clustered indicated that a fraction of this protein associates with nuclear pore complexes (NPCs) . The strong mutant phenotype, association of the protein with NPCs and genetic interaction with factors involved in RNA export provide strong evidence that Dbp5p/Rat8p plays a direct role in RNA export.

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