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