Cell Stress Chaperones, 1997 Dec, 2(4), 263 - 75 Expression of human heat shock transcription factors 1 and 2 in HeLa cells and yeast; Yuan CX et al.; We have examined reporter gene (beta-gal) expression directed by human heat shock transcription factors 1 and 2 (HSF1 and HSF2) in HeLa cells and in yeast (Saccharomyces cerevisiae) . Transcriptional activation domains of both HSFs were mapped to the C-termini using chimeric proteins containing the GAL4 DNA binding domain (GAL4-DBD) . Deletion analysis of HSF1 largely confirmed the mapping and expression pattern of activation domain 2 (AD2) previously reported by Green et al (1995) with the exception of the contribution of the oligomerization domain (hydrophobic region A) to basal repression in yeast, but not in HeLa cells . In addition, a C-terminal activation domain for HSF2 (amino acids 397 to 536) was identified by analysis in yeast . In contrast to HSF1, full length HSF2 and the isolated activation domain of HSF2 showed little activity in HeLa cells . Analysis of point mutations generated by low fidelity PCR within AD2 of HSF1 indicated that hydrophobic and charged amino acids in addition to proline, serine and threonine make critical contributions to transcriptional activity . Co-expression of GAL4-DBD fusions with AD2 of HSF1 and the C-terminal activation domain of HSF2 showed no evidence of synergism in the activation of transcription . Wild-type human HSF1 and HSF2 were both able to substitute for the endogenous yeast HSF under normal growth conditions. FEMS Microbiol Lett, 1998 Mar 1, 160(1), 61 - 7 Human transferrin receptor is active and plasma membrane-targeted in yeast; Terng HJ et al.; The human transferrin receptor, a type II plasma membrane protein which mediates iron transport in human cells, was expressed in the yeast Saccharomyces cerevisiae . The transferrin receptor synthesized by yeast cells was posttranslationally modified comparable to the native receptor with respect to glycosylation and dimer formation . The location of the expressed receptor in the yeast plasma membrane indicates that the targeting of this type II membrane protein shares similar mechanisms in yeast and mammalian cells . The yeast-expressed transferrin receptor showed binding activity towards its natural ligand, transferrin in an ELISA binding assay. Bioelectromagnetics, 1998, 19(2), 128 - 30 Ultra-wide band electromagnetic radiation does not affect UV-induced recombination and mutagenesis in yeast; Pakhomova ON et al.; Cell samples of the yeast Saccharomyces cerevisiae were exposed to 100 J/m2 of 254 nm ultraviolet (UV) radiation followed by a 30 min treatment with ultra-wide band (UWB) electromagnetic pulses . The UWB pulses (101-104 kV/m, 1.0 ns width, 165 ps rise time) were applied at the repetition rates of 0 Hz (sham), 16 Hz, or 600 Hz . The effect of exposures was evaluated from the colony-forming ability of the cells on complete and selective media and the number of aberrant colonies . The experiments established no effect of UWB exposure on the UV-induced reciprocal and non-reciprocal recombination, mutagenesis, or cell survival. J Protein Chem, 1998 Jan, 17(1), 1 - 7 Phosphatidate phosphatase--a key enzyme in glycerolipid biosynthesis . Studies on the yeast enzyme; Stark M et al.; Phosphatidate phosphatase is an important enzyme in glycerolipid biosynthesis, but difficult to purify . A purified preparation of N-ethylmaleimide-sensitive phosphatidate phosphatase from the yeast Saccharomyces cerevisiae was obtained by a five-step protocol, using chromatography on DE-53/DEAE FF, Affi-Gel Blue, hydroxyapatite, Mono-Q, and Superdex 200 . A protease-deficient yeast strain gave preparations similar to those of the wild-type strain . In exclusion chromatography, the enzyme activity of all preparations eluted at approximately the same position as albumin . However, the behavior on SDS/PAGE differed considerably among preparations, suggesting a multimeric subunit structure or degradation during purification . A 35-kDa and a 40-kDa protein band which coincided with activity were found in all preparations . Glycerol in the buffers could be excluded without rapid loss of enzyme activity, and Tris could be substituted for ammonium bicarbonate, while at least 0.6% sodium cholate in the buffers was essential. Cell, 1998 Feb 20, 92(4), 559 - 71 Sec3p is a spatial landmark for polarized secretion in budding yeast; Finger FP et al.; Exocytosis in yeast occurs at plasma membrane subdomains whose locations vary with the cell cycle, but the primary protein determinants of these sites are unknown . A functional fusion of Sec3 protein with green fluorescent protein (Sec3-GFP) localizes to the site of polarized exocytosis for each cell-cycle stage, where it colocalizes with Sec4p and Sec8p . Sec3-GFP localization is independent of secretory pathway function, of the actin and septin cytoskeletons, and of the polarity establishment proteins . We propose that Sec3p is a spatial landmark defining sites of exocytosis . Polarized secretion would result from the coupling of actin-dependent vesicle targeting with Sec3p-dependent establishment of the vesicle fusion site. Parasitol Res, 1998, 84(1), 13 - 6 Mapping the genome of Eimeria tenella: use of a yeast artificial chromosome library as the basis for a physical map; Hoogendoorn B et al.; Efforts to derive a physical map of the genome of Eimeria tenella are being made to complement genetic mapping and to assist in the production of an integrated map . A large insert library of DNA from the Houghton (H) reference strain of E . tenella has been constructed in yeast artificial chromosomes (YACs) . The library contains a tenfold E . tenella genome equivalent consisting of 3177 clones arrayed in 96-well microtitre plates and gridded on nylon membranes at 1728 clones/86 cm2 . Size fractionation of a random sample of 185 YACs revealed an average insert size of approximately 170 kb . Hybridisation of four E . tenella single-copy probes to the gridded colony filters produced around four or more positive clones . A probe representing the whole of the 1.2-Mb chromosome 2, excised from a preparative gel following pulsed-field gel electrophoresis, revealed between 80 and 100 positive clones. Nature, 1998 Feb 12, 391(6668), 660 - 6 Crystal structure of the yeast MATalpha2/MCM1/DNA ternary complex; Tan S et al.; The structure of a complex containing the homeodomain repressor protein MATalpha2 and the MADS-box transcription factor MCM1 bound to DNA has been determined by X-ray crystallography at 2.25 A resolution . It reveals the protein-protein interactions responsible for cooperative binding of MATalpha2 and MCM1 to DNA . The otherwise flexible amino-terminal extension of the MATalpha2 homeodomain forms a beta-hairpin that grips the MCM1 surface through parallel beta-strand hydrogen bonds and close-packed, predominantly hydrophobic, side chains . DNA bending induced by MCM1 brings the two proteins closer together, facilitating their interaction . An unusual feature of the complex is that an eight-amino-acid sequence adopts an alpha-helical conformation in one of two copies of the MATalpha2 monomer and a beta-strand conformation in the other . This 'chameleon' sequence of MATalpha2 may be important for recognizing natural operator sites. Eur J Biochem, 1998 Feb 1, 251(3), 812 - 20 Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein expressed in yeast; Balland A et al.; PIXY321, a human cytokine analog genetically engineered by the fusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), was expressed in yeast under the control of the alcohol dehydrogenase 2 (ADH2) promoter and the alpha-mating factor expression system . To provide the material necessary for the evaluation of PIXY321 in clinical trials, the production was scaled up to the 1200-1 scale and the PIXY321 molecule isolated by four successive steps of ion-exchange chromatography . Multiple heterogeneities, due to the presence of different patterns of glycosylation as well as multiple amino acid sequences at both N and C termini, were characterized on the purified molecule using complementary analytical techniques including electrophoresis, liquid chromatography and electrospray mass spectrometry . Four different N-terminal sequences were identified but simplified to a reproducible ratio of two sequences, the mature form and a form starting at Ala3, by adjustment of the process conditions . Molecules lacking 1-6 residues at the C-terminus were identified and their relative frequencies quantified . Amino acid modifications, such as three oxidized Met residues at positions 79, 141 and 187 and one deamidated Asn residue at position 176, were detected at low level . Microheterogeneities in glycosylation were characterized on four different sites, one located in the GM-CSF portion and three in the IL-3 portion of the molecule . The sites were shown to be differentially occupied and to carry 0-10 mannose residues according to their location in the sequence . Precise measurement of the heterogeneities at the molecular level were used to tune the process conditions and ensure reproducibility of the clinical product between lots. Eur J Biochem, 1998 Feb 1, 251(3), 775 - 80 Can monomers of yeast enolase have enzymatic activity? Kornblatt MJ, Lange R, Balny C. Using a combination of ultraviolet spectroscopy under pressure and stopped-flow kinetics under pressure, we have shown that the monomers of yeast enolase produced by hydrostatic pressure are inactive . K(eq), deltaV and deltaV for the dissociation/inactivation produced by hydrostatic pressure have been determined under various conditions . Removing the Mg2+ from enolase, either by adding EDTA or by preparing apoenzyme, displaces the equilibrium towards monomers and decreases both deltaV and deltaV . Loss of Mg2+ contributes to the negative deltaV for dissociation; this loss occurs, at least partially, in the transition state for dissociation . Both removal of Mg(II) and dissociation of the enzyme produce major changes in the intensity of the aromatic region of the CD spectrum . We propose that these changes in the CD spectra reflect changes in the conformations of the 'mobile loops' of enolase . The precise conformation of these, loops is necessary for binding Mg2+ (and, hence, for activity) and for maintaining subunit interactions. FEBS Lett, 1998 Jan 30, 422(2), 170 - 4 Carbamyl-phosphate synthetase domain of the yeast multifunctional protein Ura2 is necessary for aspartate transcarbamylase inhibition by UTP; Antonelli R et al.; In Saccharomyces cerevisiae, the first two reactions of pyrimidine biosynthesis are catalyzed by the multifunctional protein Ura2 carrying both carbamyl-phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) enzyme activities . In order to study how UTP regulates both of these activities mutant strains were constructed: one strain which expressed the Ura2 protein fused to the green fluorescent protein, and two strains expressed truncated Ura2 proteins . These strains exhibited a phenotype associated with a modified regulation of the pyrimidine pathway . Results presented in this report provide arguments in favor of a single UTP binding site located on the CPSase domain, and support a model in which ATCase activity is inhibited by UTP only when it can interact with the CPSase domain. Biochemistry, 1998 Feb 3, 37(5), 1245 - 55 Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions; Baburina I et al.; The residue C221 on pyruvate decarboxylase (EC . 4.1.1.1) from Saccharomyces cerevisiae has been shown to be the site where the substrate activation cascade is triggered {Baburina et al . (1994) Biochemistry 33, 5630-5635} and is located on the beta domain {Arjunan et al . (1996) J . Mol . Biol . 256, 590}, while the active-center thiamin diphosphate is located > 20 A away, at the interface of the alpha and gamma domains . The reactivity of all three exposed cysteines (152, 221, and 222) was examined under the influence of known activators and inhibitors . Protein chemical methods, in conjunction with {1-14C} and {3-3H} analogues of the mechanism-based inhibitor p-ClC6H4CH=CHCOCOOH, demonstrated that the holoenzyme bound approximately 2-3 atoms of tritium/atom of C-14 . However, when the labeled enzyme was subjected to trypsinization, followed by sequencing of the labeled peptide, only the tritium label was in evidence at C221, with a stoichiometry of 2 atoms of tritium/tetrameric holoenzyme . Apparently, the product of decarboxylation bonded to the enzyme survived the limited proteolysis and sequencing, but the bound 2-oxoacid was released during the protocol . Surprisingly, the C221S or C222A variants, although they still possess 20-30% specific activity compared to the wild-type enzyme, could still be inhibited by the XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds, cinnamaldehydes . Other potential nucleophilic sites for the inhibitor {C152 (the third exposed cysteine), residues D28, H114, H115, and E477 at the active center and H92 at the regulatory site} were also substituted by a nonnucleophilic side chain . All variants were still subject to inhibition by p-ClC6H4CH=CHCOCOOH, the active-center variants being inactivated even faster than the wild-type enzyme, suggesting that the active center is involved in the inactivation process . It appears that C221 is one of only two sites of interaction with such compounds (perhaps the result of a Michael addition across the C=C bond), yet the bound {1-14C}-labeled inhibitor could no longer be detected after peptide mapping at this site or at the catalytic site . Upon combining the tritiated inhibitor with {2-14C}-thiamin diphosphate, no evidence could be found for a thiamin-inhibitor-protein ternary complex, suggesting that the thiamin-bound enamine intermediate did not react further with the protein . It is likely that the second form of inhibition is at the active center, with the inhibitor cofactor-bound, which would have been released during the proteolytic protocol . Among other known activators, ketomalonate was found to react at C221 only . Glyoxalic acid, a mechanism-based inhibitor, on the other hand, could react at both the regulatory and the catalytic center . The high reactivity of C221 is consistent with it being in the thiolate form at the optimal pH of the enzyme {forming a Cys221S(-) + HHis92 ion pair; see Baburina et al . (1996) Biochemistry 35, 10249-10255, and Baburina et al . (1998) Biochemistry 37, 1235-1244} . Several additional compounds were tested as potential regulatory site-directed reagents: iodoacetate, 1,3-dibromoacetone, and 1-bromo-2-butanone . All three compounds reduced the Hill coefficient and hence appear to react at C221 . It was concluded that either substitution of C221 by a nonnucleophilic residue or large groups attached to C221 in the wild-type enzyme lead to a distortion of domain interactions, interactions which are required for both optimal activity and substrate activation. Science, 1998 Feb 13, 279(5353), 1045 - 7 Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint; Kim SH et al.; Mad2 is a component of the spindle checkpoint, which delays the onset of anaphase until all chromosomes are attached to the spindle . Mad2 formed a complex with Slp1, a WD (tryptophan-aspartic acid)-repeat protein essential for the onset of anaphase . When the physical interaction between the two proteins was disrupted, the spindle checkpoint was no longer functional . Post-anaphase events such as chromosome decondensation and the next round of DNA replication were not delayed even when the spindle assembly was incomplete . This relief of dependence appears to be a result of deregulation of ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex. Science, 1998 Feb 13, 279(5353), 1041 - 4 Budding yeast Cdc20: a target of the spindle checkpoint; Hwang LH et al.; The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis . In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis . Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle . The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2 . Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA . Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint. J Biol Chem, 1998 Feb 6, 273(6), 3381 - 7 In vitro reconstitution of glucose-induced targeting of fructose-1, 6-bisphosphatase into the vacuole in semi-intact yeast cells; Shieh HL et al.; Fructose-1,6-bisphosphatase (FBPase), the key enzyme in gluconeogenesis in the yeast Saccharomyces cerevisiae, is induced when cells are grown in medium containing poor carbon sources . FBPase is targeted from the cytosol to the vacuole for degradation when glucose-starved yeast cells are replenished with fresh glucose . In this study, we report the reconstitution of the glucose-induced import of FBPase into the vacuole in semi-intact yeast cells using radiolabeled FBPase, an ATP regenerating system and cytosol . The import of FBPase was defined as the fraction of the FBPase that was sequestered inside a membrane-sealed compartment . FBPase import requires ATP hydrolysis and is stimulated by cytosolic proteins . Furthermore, the import of FBPase is a saturable process . FBPase import is low in the glucose-starved cells and is stimulated in the glucose-replenished cells . FBPase accumulates to a higher level in the pep4 cell, suggesting that FBPase is targeted to the vacuole for degradation . Indirect immunofluorescence microscopy studies demonstrate that the imported FBPase is localized to the vacuole in the permeabilized cells . Thus, the glucose-induced targeting of FBPase into the vacuole can be reproduced in our in vitro system. J Biol Chem, 1998 Feb 6, 273(6), 3351 - 7 Yeast Clk-1 homologue (Coq7/Cat5) is a mitochondrial protein in coenzyme Q synthesis; Jonassen T et al.; Mutations in the clk-1 gene result in slower development and increased life span in Caenorhabditis elegans . The Saccharomyces cerevisiae homologue COQ7/CAT5 is essential for several metabolic pathways including ubiquinone biosynthesis, respiration, and gluconeogenic gene activation . We show here that Coq7p/Cat5p is a mitochondrial inner membrane protein directly involved in ubiquinone biosynthesis, and that the defect in gluconeogenic gene activation in coq7/cat5 null mutants is a general consequence of a defect in respiration . These results obtained in the yeast model suggest that the effects on development and life span in C . elegans clk-1 mutants may relate to changes in the amount of ubiquinone, an essential electron transport component and a lipid soluble antioxidant. Genes Dev, 1998 Jan 15, 12(2), 219 - 32 Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci; Enomoto S et al.; CAC1/RLF2 encodes the largest subunit of chromatin assembly factor I (CAF-I), a complex that assembles newly synthesized histones onto recently replicated DNA in vitro . In vivo, cac1/rlf2 mutants are defective in telomeric silencing and mislocalize Rap1p, a telomere-binding protein . Here, we report that in cells lacking CAF-I the silent mating loci are derepressed partially . MATa cac1 cells exhibit an unusual response to alpha-factor: They arrest and form mating projections (shmoos) initially, but are unable to sustain the arrest state, giving rise to clusters of shmooing cells . cac1 MATa HMLa HMRa strains do not form these shmoo clusters, indicating that derepression of HMLalpha causes the shmoo cluster phenotype in cac1 cells . When SIR3 is reintroduced into sir1 sir3 cells, HML remains derepressed indicating that SIR1 is required for the re-establishment of silencing at HML . In contrast, when SIR3 is reintroduced into cac1 sir3 cells, silencing is restored to HML, indicating that CAF-I is not required for the re-establishment of silencing . Loss of the other CAF-I subunits (Cac2p and Cac3p/Msi1p) also results in the shmoo cluster phenotype, implying that loss of CAF-I activity gives rise to this unstable repression of HML . Strains carrying certain mutations in the amino terminus of histone H4 and strains with limiting amounts of Sir2p or Sir3p also form shmoo clusters, implying that the shmoo cluster phenotype is indicative of defects in maintenance of the structural integrity of silent chromatin . MATa cac- sir1 double mutants have a synergistic mating defect, suggesting that the two silencing mechanisms, establishment and maintenance, function cooperatively . We propose a model to explain the distinctions between the establishment and the maintenance of silent chromatin. Genes Dev, 1998 Jan 15, 12(2), 198 - 207 The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast grr1p; Ruegger M et al.; Genetic analysis in Arabidopsis has led to the identification of several genes that are required for auxin response . One of these genes, AXR1, encodes a protein related to yeast Aos1p, a protein that functions to activate the ubiquitin-related protein Smt3p . Here we report the identification of a new gene called TRANSPORT INHIBITOR RESPONSE 1 (TIR1) . The tir1 mutants are deficient in a variety of auxin-regulated growth processes including hypocotyl elongation and lateral root formation . These results indicate that TIR1 is also required for normal response to auxin . Further, mutations in TIR1 display a synergistic interaction with mutations in AXR1, suggesting that the two genes function in overlapping pathways . The TIR1 protein contains a series of leucine-rich repeats and a recently identified motif called an F box . Sequence comparisons indicate that TIR1 is related to the yeast protein Grr1p and the human protein SKP2 . Because Grr1p and other F-box proteins have been implicated in ubiquitin-mediated processes, we speculate that auxin response depends on the modification of a key regulatory protein(s) by ubiquitin or a ubiquitin-related protein. Mol Cell Biol, 1998 Mar, 18(3), 1701 - 10 Cloning and biochemical characterization of TAF-172, a human homolog of yeast Mot1; Chicca JJ 2nd et al.; The TATA binding protein (TBP) is a central component of the eukaryotic transcriptional machinery and is the target of positive and negative transcriptional regulators . Here we describe the cloning and biochemical characterization of an abundant human TBP-associated factor (TAF-172) which is homologous to the yeast Mot1 protein and a member of the larger Snf2/Swi2 family of DNA-targeted ATPases . Like Mot1, TAF-172 binds to the conserved core of TBP and uses the energy of ATP hydrolysis to dissociate TBP from DNA (ADI activity) . Interestingly, ATP also causes TAF-172 to dissociate from TBP, which has not been previously observed with Mot1 . Unlike Mot1, TAF-172 requires both TBP and DNA for maximal (approximately 100-fold) ATPase activation . TAF-172 inhibits TBP-driven RNA polymerase II and III transcription but does not appear to affect transcription driven by TBP-TAF complexes . As it does with Mot1, TFIIA reverses TAF-172-mediated repression of TBP . Together, these findings suggest that human TAF-172 is the functional homolog of yeast Mot1 and uses the energy of ATP hydrolysis to remove TBP (but apparently not TBP-TAF complexes) from DNA. Mol Cell Biol, 1998 Mar, 18(3), 1534 - 43 Mutations in the yeast KEX2 gene cause a Vma(-)-like phenotype: a possible role for the Kex2 endoprotease in vacuolar acidification; Oluwatosin YE et al.; Mutants of Saccharomyces cerevisiae that lack vacuolar proton-translocating ATPase (V-ATPase) activity show a well-defined set of Vma- (stands for vacuolar membrane ATPase activity) phenotypes that include pH-conditional growth, increased calcium sensitivity, and the inability to grow on nonfermentable carbon sources . By screening based on these phenotypes and the inability of vma mutants to accumulate the lysosomotropic dye quinacrine in their vacuoles, five new vma complementation groups (vma41 to vma45) were identified . The VMA45 gene was cloned by complementation of the pH-conditional growth of the vma45-1 mutant strain and shown to be allelic to the previously characterized KEX2 gene, which encodes a serine endoprotease localized to the late Golgi compartment . Both vma45-1 mutants and kex2 null mutants exhibit the full range of Vma- growth phenotypes and show no vacuolar accumulation of quinacrine, indicating loss of vacuolar acidification in vivo . However, immunoprecipitation of the V-ATPase from both strains under nondenaturing conditions revealed no defect in assembly of the enzyme, vacuolar vesicles isolated from a kex2 null mutant showed levels of V-ATPase activity and proton pumping comparable to those of wild-type cells, and the V-ATPase complex purified from kex2 null mutants was structurally indistinguishable from that of wild-type cells . The results suggest that kex2 mutations exert an inhibitory effect on the V-ATPase in the intact cell but that the ATPase is present in the mutant strains in a fully assembled state, potentially capable of full enzymatic activity . This is the first time a mutation of this type has been identified. Mol Cell Biol, 1998 Mar, 18(3), 1449 - 58 Identification and functional characterization of a novel nuclear localization signal present in the yeast Nab2 poly(A)+ RNA binding protein; Truant R et al.; The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin alpha/importin alpha, which acts as the NLS receptor, and karyopherin beta1/importin beta, which binds karyopherin alpha and mediates the nuclear import of the resultant ternary complex . Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified . In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described . The M9 NLS binds a transport factor related to karyopherin beta1, termed karyopherin beta2 or transportin, and does not require a karyopherin alpha-like adapter protein . A yeast homolog of karyopherin beta2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p . Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs . Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS . In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p . Surprisingly, this in vitro assay also revealed that human karyopherin beta1, but not the Kap104p homolog karyopherin beta2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin alpha . These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences. Mol Cell Biol, 1998 Mar, 18(3), 1284 - 95 The C terminus of the major yeast telomere binding protein Rap1p enhances telomere formation; Ray A et al.; The telomeres of most organisms consist of short repeated sequences that can be elongated by telomerase, a reverse transcriptase complex that contains its own RNA template for the synthesis of telomere repeats . In Saccharomyces cerevisiae, the RAP1 gene encodes the major telomere binding protein Rap1p . Here we use a quantitative telomere formation assay to demonstrate that Rap1p C termini can enhance telomere formation more than 30-fold when they are located at internal sites . This stimulation is distinct from protection from degradation . Enhancement of formation required the gene for telomerase RNA but not Sir1p, Sir2p, Sir3p, Sir4p, Tel1p, or the Rif1p binding site in the Raplp C terminus . Our data suggest that Rap1p C termini enhance telomere formation by attracting or increasing the activity of telomerase near telomeres . Earlier work suggests that Rap1p molecules at the chromosome terminus inhibit the elongation of long telomeres by blocking the access of telomerase . Our results suggest a model where a balance between internal Rap1p increasing telomerase activity and Rap1p at the termini of long telomeres controlling telomerase access maintains telomeres at a constant length. Appl Microbiol Biotechnol, 1998 Jan, 49(1), 66 - 71 Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast; Polakowski T et al.; The enzyme 3-hydroxy-3-methylglutaryl-co-enzyme-A (HMG-CoA) reductase is known as the rate limiting enzyme in early sterol biosynthesis in eukaryotic cells . To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e . amino acids 1-552), was constructed and overexpressed in this yeast . The transformed strains accumulated large amounts of the sterol precursor squalene, while the levels of ergosterol and a number of other sterol compounds were only slightly elevated . These findings suggest that HMG-CoA reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this pathway beyond the sterol precursor squalene. Nat Biotechnol, 1998 Feb, 16(2), 186 - 9 Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast; Duport C et al.; The first two steps of the steroidogenic pathway were reproduced in Saccharomyces cerevisiae . Engineering of sterol biosynthesis by disruption of the delta 22-desaturase gene and introduction of the Arabidopsis thaliana delta 7-reductase activity and coexpression of bovine side chain cleavage cytochrome P450, adrenodoxin, and adrenodoxin reductase, lead to pregnenolone biosynthesis from a simple carbon source . Following additional coexpression of human 3 beta-hydroxysteroid dehydrogenase/isomerase, pregnenolone is further metabolized to progesterone . Steroid formation appears to be coupled to yeast sterol biosynthesis. Yeast, 1998 Jan 30, 14(2), 171 - 80 Functional comparison of the yeast scERV1 and scERV2 genes; Stein G et al.; The yeast scERV1 gene is the first representative of a new emerging gene family . Its gene product is essential for the yeast cell and is involved in the biogenesis of mitochondria and the regulation of the cell cycle . Recently the general importance of the gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue . The homologous mammalian ALR (augmenter of liver regeneration) genes from man, mouse and rat are important for different developmental stages of the organism as for example in spermatogenesis and the regeneration of damaged liver organs . Latest research identified an intron with an unusual 3' branch site in the 5' region of the yeast scERV1 gene . Analysis of the now available complete genome sequence from Saccharomyces cerevisiae identified a second yeast gene with homologies to scERV1 on chromosome 16 . The corresponding gene product has a length of 196 amino acids similar to the 189 residues of the scERV1 protein and exhibits 30% identical amino acid residues in the highly conserved carboxy-terminal part of the polypeptides . Because of the structural similarities the new gene will be termed scERV2 from now on . For the scERV1 gene product it has just been shown that it is associated with yeast mitochondria . Analysis of the amino-terminal part of the putative scERV2 protein also identifies a typical leader sequence for import into mitochondria . The comparison of cDNA and genomic DNA from the scERV2 gene shows that no intron is present in this gene . To investigate the functional relation between the two yeast genes disruption experiments and complementation studies of mutants from scERV1 were performed . In addition the expression of messenger RNA under 15 different growth conditions was investigated by detailed Northern hybridization studies . Both genes show a complex and distinct expression pattern for their transcripts and are highly regulated under different physiological conditions . Moreover correct and efficient splicing of the transcript from the scERV1 gene was found to vary with the physiological state of the yeast cell, as further verified by reverse transcription-polymerase chain reaction analysis of transcripts from galactose-grown yeast cells. Yeast, 1998 Jan 30, 14(2), 147 - 60 Flow cytometry and cell sorting for yeast viability assessment and cell selection; Deere D et al.; Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities . Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable . The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h . We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY) . PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use . Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox . The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability . Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells . Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments . The Ox assay was in agreement with CY and PI for live/heat-killed mixtures . However, for re-hydrated HADY, Ox stained a significantly (P < or = 0.05) higher proportion of cells than did PI. Yeast, 1998 Jan 30, 14(2), 103 - 14 Mutations in five loci affecting GAP1-independent uptake of neutral amino acids in yeast; Jorgensen MU et al.; In order to identify genes involved in uptake of isoleucine, leucine and valine in Saccharomyces cerevisiae we isolated mutants that, on a complex medium, were sensitive to an inhibitor of the biosynthesis of the branched-chain amino acids . Mutants that in a secondary screen showed reduced uptake of isoleucine, leucine and valine when growing in synthetic complete medium were further characterized . Genetic analysis identified five loci, named ssy1 through ssy5 . ssy2 corresponds to the previously characterized bap1 mutation, which we recently have found to be allelic to stp1 . ssy1, ssy3 and ssy5 exhibit a reduced uptake of phenylalanine, methionine and threonine, as well . Furthermore, they are resistant to several neutral amino acid analogs . ssy4 only affects uptake of few neutral amino acids and is as sensitive as the wild type to the amino acid analogs tested . It was previously found that a C-terminal truncation of 29 codons of BAP2, which encodes a branched-chain amino acid permease, results in increased uptake of the branched-chain amino acids . We find epistasis of the C-terminally truncated BAP2 gene over the ssy4 mutation, while the other ssy mutations are epistatic over the truncated BAP2 gene . SSY1, SSY3 and SSY5 were cloned from a low-copy genomic library by complementation of the mutants . The SSY3 gene and the SSY5 gene show no significant homology to any sequence in the databases . SSY1 is a member of the major family of genes encoding amino acid permeases in yeast . We discuss possible roles of Ssy1p in amino acid uptake. Yeast, 1998 Jan 15, 14(1), 25 - 35 Distribution of the flocculation protein, flop, at the cell surface during yeast growth: the availability of flop determines the flocculation level; Bony M et al.; The yeast FLO genes encode cell surface proteins which are expected to play a major role in the control of flocculation . We have assessed the availability of the Flo proteins at the cell surface during the growth of two flocculent strains, ABXL-1D (FLO1) and STX347-1D (FLO5) using immunological approaches, enzyme-linked immunosorbent assays and immunofluorescence . Our data show that they are not permanently present at the cell surface but that their amount increases during growth . With both strains the flocculation level is tightly correlated to the amount of Flop antigen detected, suggesting that it is the availability of the Flo proteins at the cell surface which determines the flocculation level . Our data are consistent with the idea that the Flo proteins correspond to the flocculation lectins . The differences of flocculation pattern among strains could originate from variations in the regulation of the expression of the FLO genes . Monitoring of the distribution of the Flo proteins during cellular development revealed that they are incorporated essentially in the cell wall of growing buds . Incorporation of the Flo proteins in the cell wall displays a highly polarized aspect, at the bud tip and at the mother-daughter neck junction, which can persist in mature cells . Such a localization could be relevant to constraints of the cell wall incorporation of the mannoproteins . Depending on the regulation of Flop expression and on the incorporation of the proteins in the cell wall, a yeast population can be highly heterogeneous in Flo protein equipment. Biochemistry, 1998 Feb 17, 37(7), 1769 - 76 X-ray crystal structure of the yeast Kar3 motor domain complexed with Mg.ADP to 2.3 A resolution; Gulick AM et al.; The kinesin family of motor proteins, which contain a conserved motor domain of approximately 350 amino acids, generate movement against microtubules . Over 90 members of this family have been identified, including motors that move toward the minus or plus end of microtubules . The Kar3 protein from Saccharomyces cerevisiae is a minus end-directed kinesin family member that is involved in both nuclear fusion, or karyogamy, and mitosis . The Kar3 protein is 729 residues in length with the motor domain located in the C-terminal 347 residues . Recently, the three-dimensional structures of two kinesin family members have been reported . These structures include the motor domains of the plus end-directed kinesin heavy chain {Kull, F . J., et al . (1996) Nature 380, 550-555} and the minus end-directed Ncd {Sablin, E . P., et al . (1996) Nature 380, 555-559} . We now report the structure of the Kar3 protein complexed with Mg.ADP obtained from crystallographic data to 2.3 A . The structure is similar to those of the earlier kinesin family members, but shows differences as well, most notably in the length of helix alpha 4, a helix which is believed to be involved in conformational changes during the hydrolysis cycle. Oncogene, 1998 Feb 19, 16(7), 873 - 81 Identification of homo- and heteromeric interactions between members of the breast carcinoma-associated D52 protein family using the yeast two-hybrid system; Byrne JA et al.; The hD52 gene was originally identified through its elevated expression level in human breast carcinoma . Cloning of D52 homologues from other species has indicated that D52 may play roles in calcium-mediated signal transduction and cell proliferation . Two human homologues of hD52, hD53 and hD54, have also been identified, demonstrating the existence of a novel gene/protein family . Since D52-like protein sequences are all predicted to contain a coiled-coil domain, we used the yeast two-hybrid system and glutathione S-transferase pull-down assays to investigate whether homo- and/or heteromeric interactions occur between D52-like proteins . Analyses of yeast strains co-transfected with paired D52-like constructs indicated that D52-like fusion proteins interact in homo- and heteromeric fashions through their predicted coiled-coil domains . Similarly, extensive two-hybrid screenings of a human breast carcinoma expression library identified hD53 and hD52 as potential interactors for both hD52 and hD53 baits . Thus, D52-like proteins appear to exert and/or regulate their activities through specific interactions with other D52-like proteins, which in turn may be intrinsic to potential roles of these molecules in controlling cell proliferation. Curr Opin Cell Biol, 1998 Feb, 10(1), 112 - 6 Control of reorganization of the actin cytoskeleton by Rho family small GTP-binding proteins in yeast; Tanaka K et al.; Accumulating evidence indicates that Rho family small GTP-binding proteins regulate reorganization of the actin cytoskeleton . There are members of the Rho family in the budding yeast Saccharomyces cerevisiae, in which powerful molecular genetical approaches are applicable . Recent identification of regulators and targets of the Rho family members has enhanced our understanding of the regulation and modes of action of Rho family members in reorganization of the actin cytoskeleton. J Med Chem, 1998 Feb 12, 41(4), 540 - 54 29-Methylidene-2,3-oxidosqualene derivatives as stereospecific mechanism-based inhibitors of liver and yeast oxidosqualene cyclase; Ceruti M et al.; Two pairs of isomers (18Z)- (8), (18E)-29-methylidene-2,3-oxidohexanorsqualene (21), and (18Z)- (31), (18E)-29-methylidene-2,3-oxidosqualene (34), have been obtained in a fully stereospecific manner, as inhibitors of rat and yeast oxidosqualene cyclase . A new method for the synthesis of C22 squalene aldehyde 2,3-epoxide is reported, as well as that of other 19-modified 2,3-oxidosqualene analogues . We found that the activity is the opposite in the two series: the (E)-hexanormethylidene 21 and the (Z)-methylidene 31 are potent and irreversible inhibitors of oxidosqualene cyclase, while (Z)-hexanormethylidene 8 and (E)-methylidene 34 are almost completely inactive . Reduction of the 18,19-double bond, such as in 39, eliminates the activity, while removal of both of the 19-linked groups such as in heptanor derivative 40 greatly reduces inhibition of the enzyme . (E)-Hexanormethylidene 21 results the first irreversible inhibitor of the series toward the yeast enzyme. Plant Mol Biol, 1998 Mar, 36(4), 601 - 12 Expression of fission yeast cdc25 alters the frequency of lateral root formation in transgenic tobacco; McKibbin RS et al.; Lateral root formation was examined following the expression of a fission yeast mitotic regulator gene, cdc25, under the control of a tetracycline-inducible promoter in cultured roots of tobacco . Over expression of cdc25 in fission yeast results in premature cell division at a reduced cell size . Our aim was to examine whether cdc25 expression would affect cell size in the tobacco roots, and what effect this would have on lateral root morphogenesis . Transgene integration was confirmed by Southern blotting; it was inherited as a dominant Mendelian trait . Conditions for optimal expression, determined using plants transformed with gus under the control of the same promoter, were: addition of tetracycline (5 micrograms/ml) every 72 h, to cultured roots in Murashige-Skoog liquid medium in darkness at 27 degrees C . After the addition of tetracycline, cdc25 transcripts were detected using RT-PCR, initially after 48 h, and more strongly after 72 h . Appearance of cdc25 transcripts was followed by major changes in the roots . Compared with controls, lateral root primordia were initiated more frequently, were significantly smaller and comprised smaller cells at mitosis . However, cdc25 expression did not perturb normal development of the lateral roots . The data are consistent with cdc25 expression leading to a greater frequency of lateral root primordium formation and establishing a new threshold size for cell division in the primordia which was then maintained throughout subsequent development. Plant Mol Biol, 1998 Mar, 36(4), 529 - 39 Characterization of a Sorghum bicolor gene family encoding putative protein kinases with a high similarity to the yeast SNF1 protein kinase; Annen F et al.; C4 photosynthesis is functionally dependent on metabolic interactions between mesophyll and bundle-sheath cells . Although the C4 cycle is biochemically well understood many aspects of the regulation of enzyme activities, gene expression and cell differentiation are elusive . Protein kinases are likely involved in these regulatory processes providing links to hormonal, metabolic and developmental signal transduction pathways . We have identified several protein kinases that are differentially expressed in mesophyll and bundle-sheath cells of the C4 plant Sorghum bicolor . Here we describe the characterization of two putative protein kinases that show high similarity to the SNF1/AMPK family of protein serine/threonine kinases . The mRNA of both kinases accumulates to much higher levels in mesophyll cells than in the bundle-sheath and can also be detected in root tissue . Complementation experiments with a snf1 mutant of Saccharomyces cerevisiae indicate that the S . bicolor protein kinase SNFL1 does not represent a functional homologue of the yeast SNF1 protein kinase. Acta Med Austriaca, 1997, 24(5), 185 - 7 {Effect of chromium yeast and chromium picolinate on body composition of obese, non-diabetic patients during and after a formula diet}; Bahadori B et al.; The objective of this study was to assess the effects of chromium yeast and chromium picolinate on lean body mass during and after weight reduction with a very-low-calorie diet . 36 obese (BMI 33.7 +/- 5.4 kg/m2), non-diabetic patients aged 45 +/- 6 years undergoing a 8-week very-low-calorie diet followed by a 18-week maintenance period . During the whole 26 week treatment period subjects received either placebo or chromium yeast (200 micrograms/d) or chromium-picolinate (200 micrograms/d) in a double-blind manner . Body weight was measured as BMI and body composition after calculation from skinfold thickness . As a result all three groups showed comparable weight loss after 8 and 26 weeks . Lean body mass was reduced in all groups after 8 weeks . However, after 26 weeks chromium picolinate supplemented subjects showed increased lean body mass (p < 0.029) whereas the other treatment groups still had reduced lean body mass . Chromium picolinate, but not chromium yeast, is able to increase lean body mass in obese patients in the maintenance period after a very-low-calorie diet without counteracting the weight loss achieved. Science, 1998 Feb 6, 279(5352), 853 - 6 Expansion and length-dependent fragility of CTG repeats in yeast; Freudenreich CH et al.; Expansion of DNA trinucleotide repeats (TNRs) is the causative mutation in a growing number of human genetic diseases . Large expansions of a CTG tract were obtained and shown by genetic and physical assays to be length-dependent sites of chromosome breakage in Saccharomyces cerevisiae . Deletion of RAD27, which encodes a nuclease involved in Okazaki fragment processing, caused length-dependent destabilization of CTG tracts and a substantial increase in expansion frequency . The genetic assay described here can be used to evaluate other factors that induce TNR expansion or chromosome fragility in humans. J Biol Chem, 1998 Jan 30, 273(5), 2993 - 3002 Mutational studies with Atp12p, a protein required for assembly of the mitochondrial F1-ATPase in yeast . Identification of domains important for Atp12p function and oligomerization; Wang ZG et al.; The Atp12p protein of Saccharomyces cerevisiae is required for assembly of the F1 moiety of the mitochondrial ATP synthase . The current work has used mutant forms of Atp12p in an effort to learn about amino acids and/or domains that are important for the action of the protein . In one set of studies, the mutant atp12 genes were cloned and sequenced from 13 independent isolates of chemically mutagenized yeast . Of the 10 different mutant alleles that were identified, 9 (8 nonsense and 1 frameshift) lead to the early termination of the protein . A single missense mutation that substitutes lysine for Glu-289 was identified in two of the atp12 strains . Analysis of several Atp12p variants, each with different substitutions at Glu-289, showed that the functional activity of Atp12p is compromised when non-acidic residues are introduced at position 289 in the sequence . In other work, deletion analysis led to the assignment of two domains in Atp12p; the functional domain of the protein was mapped to the sequence between Gln-181 and Val-306, and a structural domain (Asp-307 through Gln-325) was identified that confers Atp12p the ability to oligomerize with other proteins in mitochondria. J Cell Biol, 1998 Jan 26, 140(2), 377 - 90 Kar9p is a novel cortical protein required for cytoplasmic microtubule orientation in yeast; Miller RK et al.; kar9 was originally identified as a bilateral karyogamy mutant, in which the two zygotic nuclei remained widely separated and the cytoplasmic microtubules were misoriented (Kurihara, L.J., C.T . Beh, M . Latterich, R . Schekman, and M.D . Rose . 1994 . J . Cell Biol . 126:911-923.) . We now report a general defect in nuclear migration and microtubule orientation in kar9 mutants . KAR9 encodes a novel 74-kD protein that is not essential for life . The kar9 mitotic defect was similar to mutations in dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5 . kar9Delta dhc1Delta, kar9Delta jnm1Delta, and kar9Delta act5Delta double mutants were synthetically lethal, suggesting that these genes function in partially redundant pathways to carry out nuclear migration . A functional GFP-Kar9p fusion protein localized to a single dot at the tip of the shmoo projection . In mitotic cells, GFP-Kar9p localized to a cortical dot with both mother-daughter asymmetry and cell cycle dependence . In small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud . In telophase and G1 unbudded cells, no localization was observed . By indirect immunofluorescence, cytoplasmic microtubules intersected the GFP-Kar9p dot . Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent . We propose that Kar9p is a component of a cortical adaptor complex that orients cytoplasmic microtubules. J Cell Biol, 1998 Jan 26, 140(2), 355 - 66 Sequential assembly of myosin II, an IQGAP-like protein, and filamentous actin to a ring structure involved in budding yeast cytokinesis; Lippincott J et al.; We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs . Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle . Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother-bud junction after the initiation of anaphase . This ring contains filamentous actin and Myo1p, a myosin II homologue . In vivo observation with green fluorescent protein-tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile . These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring . The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis . The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring . The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs. J Cell Biol, 1998 Jan 26, 140(2), 247 - 58 A novel fission yeast gene, tht1+, is required for the fusion of nuclear envelopes during karyogamy; Tange Y et al.; We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked . The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci . The tht1(+) gene was cloned and sequenced . Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein . The tht1(+) gene is dispensable for vegetative growth and expressed only in conjugating cells . Tht1p is a glycoprotein susceptible to endoglycosilase H digestion . Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function . A protease protection assay indicated that the COOH terminus is cytoplasmic . Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei. EMBO J, 1998 Jan 15, 17(2), 565 - 74 Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing; O'Keefe RT et al.; The U5 snRNA loop 1 interacts with the 5' exon before the first step of pre-mRNA splicing and with the 5' and 3' exons following the first step . These U5-exon interactions are proposed to hold the exons in the correct orientation for the second step of splicing . Reconstitution of U5 snRNPs in vitro indicated that U5 loop 1-5' exon interactions are not necessary for the first catalytic step of splicing but are critical for the second step in yeast spliceosomes . We systematically made deletion and insertion mutations in loop 1 then monitored splicing activity and loop-exon interactions by cross-linking . Single nucleotide deletions or insertions in loop 1 permitted both steps of splicing . Larger insertions or deletions allowed the first step but progressively inhibited the second step . Analysis of selected loop 1 insertions and deletions by cross-linking revealed that inhibition of the second catalytic step resulted from misalignment of the 5' and 3' exons . These data indicate that the size of loop 1 is critical for proper alignment of the exons for the second catalytic step of splicing and that the 3' exon is positioned on loop 1 independently of the 5' exon. EMBO J, 1998 Jan 15, 17(2), 498 - 506 Budding yeast RSI1/APC2, a novel gene necessary for initiation of anaphase, encodes an APC subunit; Kramer KM et al.; SIC1 is a non-essential gene encoding a CDK inhibitor of Cdc28-Clb kinase activity . Sic1p is involved in both mitotic exit and the timing of DNA synthesis . To identify other genes involved in controlling Clb-kinase activity, we have undertaken a genetic screen for mutations which render SIC1 essential . Here we describe a gene we have identified by this means, RSI1/APC2 . Temperature-sensitive rsi1/apc2 mutants arrest in metaphase and are unable to degrade Clb2p, suggesting that Rsi1p/Apc2p is associated with the anaphase promoting complex (APC) . This is an E3 ubiquitin-ligase that controls anaphase initiation through degradation of Pds1p and mitotic exit via degradation of Clb cyclins . Indeed, the anaphase block in rsi1/apc2 temperature-sensitive mutants is overcome by removal of PDS1, consistent with Rsi1p/Apc2p being part of the APC . In addition, like our rsi1/apc2 mutations, cdc23-1, encoding a known APC subunit, is also lethal with sic1Delta . Thus SIC1 clearly becomes essential when APC function is compromised . Finally, we find that Rsi1p/Apc2p co-immunoprecipitates with Cdc23p . Taken together, our results suggest that RSI1/APC2 is a subunit of APC. EMBO J, 1998 Jan 2, 17(1), 140 - 8 pmp1+, a suppressor of calcineurin deficiency, encodes a novel MAP kinase phosphatase in fission yeast; Sugiura R et al.; Calcineurin is a highly conserved and ubiquitously expressed Ca2+- and calmodulin-dependent protein phosphatase . The in vivo role of calcineurin, however, is not fully understood . Here, we show that disruption of the calcineurin gene (ppb1(+)) in fission yeast results in a drastic chloride ion (Cl-)-sensitive growth defect and that a high copy number of a novel gene pmp1(+) suppresses this defect . pmp1(+) encodes a phosphatase, most closely related to mitogen-activated protein (MAP) kinase phosphatases of the CL100/MKP-1 family . Pmp1 and calcineurin share an essential function in Cl- homeostasis, cytokinesis and cell viability . Pmp1 phosphatase dephosphorylates Pmk1, the third MAP kinase in fission yeast, in vitro and in vivo, and is bound to Pmk1 in vivo, strongly suggesting that Pmp1 negatively regulates Pmk1 MAP kinase by direct dephosphorylation . Consistently, the deletion of pmk1(+) suppresses the Cl--sensitive growth defect of ppb1 null . Thus, calcineurin and the Pmk1 MAP kinase pathway may play antagonistic functional roles in the Cl- homeostasis. EMBO J, 1998 Jan 2, 17(1), 113 - 26 Two syntaxin homologues in the TGN/endosomal system of yeast; Holthuis JC et al.; Intracellular membrane traffic is thought to be regulated in part by SNAREs, integral membrane proteins on transport vesicles (v-SNAREs) and target organelles (t-SNAREs) that bind to each other and mediate bilayer fusion . All known SNARE-mediated fusion events involve a member of the syntaxin family of t-SNAREs . Sequence comparisons identify eight such proteins encoded in the yeast genome, of which six have been characterized . We describe here the remaining two, Tlg1p and Tlg2p . These have the expected biochemical properties of t-SNAREs, and are located in separable compartments which correspond to a putative early endosome and the yeast equivalent of the TGN, respectively . They co-precipitate with the v-SNARE Vti1p, which is implicated in Golgi-endosome traffic and, remarkably, binds to five different syntaxins . Tlg1p also binds the plasma membrane v-SNARE Snc1p . Both Tlg1p and Tlg2p are required for efficient endocytosis and to maintain normal levels of TGN proteins . However, neither is required for intra-Golgi traffic . Since no further syntaxins have been identified in yeast, this implies that the Golgi apparatus can function with a single syntaxin, Sed5p. Biochem J, 1998 Jan 15, 329 ( Pt 2), 359 - 67 Influence of N-terminal sequence variation on the sorting of major adenylate kinase to the mitochondrial intermembrane space in yeast; Bandlow W et al.; Major adenylate kinase (Aky2p) from yeast has no cleavable presequence and occurs in identical form in the mitochondrial intermembrane space (6-8%) and in the cytoplasm (approx . 90%) . To identify the signal(s) on Aky2p that might be required for mitochondrial import, the N-terminal region was examined . The N-terminus of Aky2p can guide at least two cytoplasmic passengers, dihydrofolate reductase from mouse and UMP kinase (Ura6p) from yeast, to the intermembrane space in vivo, showing that the N-terminus harbours import information . In contrast, deletion of the eight N-terminal amino acid residues or the introduction of two compensating frameshifts into this segment does not abolish translocation into the organelle's intermembrane space . Thus internal targeting and sorting information must be present in Aky2p as well . Neither a pronounced amphiphilic alpha-helical moment nor positive charges in the N-terminal region is a necessary prerequisite for Aky2p to reach the intermembrane space . Even a surplus of negative charges in mutant N-termini does not impede basal import into the correct submitochondrial compartment . The potential to form an amphipathic alpha-helical structure of five to eight residues close to the N-terminus significantly improves import efficiency, whereas extension of this amphipathic structure, e.g . by replacing it with the homologous segment of Aky3p, a mitochondrial matrix protein from yeast, leads to misdirection of the chimaera to the matrix compartment . This shows that the topogenic N-terminal signal of Aky3p is dominant over the presumptive internal intermembrane space-targeting signal of Aky2p and argues that the sorting of wild-type Aky2p to the intermembrane space is not due to the presence in the protein of a specific sorting sequence for the intermembrane space, but rather is the consequence of being imported but not being sorted to the inner compartment . Some Aky2 mutant proteins are susceptible to proteolysis in the cytoplasm, indicating incorrect folding . They are nevertheless efficiently rescued by uptake into mitochondria, suggesting a negative correlation between folding velocity (or folding stability) and efficiency of import. Hum Mol Genet, 1998 Jan, 7(1), 69 - 74 Expansions of CAG repeat tracts are frequent in a yeast mutant defective in Okazaki fragment maturation; Schweitzer JK et al.; To understand the causes of CAG repeat tract changes that occur in the passage of human disease alleles, we are studying the effect of replication and repair mutations on CAG repeat tracts embedded in a yeast chromosome . In this report, we examine the effect of a mutation in the RTH1/RAD27 gene encoding a deoxyribonuclease needed for removal of excess nucleotides at the 5'-end of Okazaki fragments . Deletion of the RTH1/RAD27 gene has two effects on CAG tracts . First, the rth1/rad27 mutation destabilizes CAG tracts . Second, although most tract length changes in wild-type yeast cells are tract contractions, approximately half of the changes that occur as a result of the rth1/rad27 mutation are expansions of one or more repeat units . These results support the hypothesis that tract expansions that occur during passage of human disease alleles bearing expanded CAG tracts result from excess DNA synthesis on the lagging strand of replication. Mycoses, 1997 Nov, 40(7-8), 303 - 8 Detection of antibodies and delayed dermal hypersensitivity with different lots of Blastomyces dermatitidis yeast lysate antigen: stability and specificity evaluations; Wakamoto A et al.; Comparative evaluations were performed to assess the stability, sensitivity and specificity of eight lots of yeast lysate antigen prepared from a Blastomyces dermatitidis dog isolate (T-58) . These antigens were prepared during the period from 1989 to 1995 . The lysates were used in an ELISA for the detection of antibodies in serum specimens from dogs with blastomycosis and histoplasmosis . In order to evaluate the ability of the lysates to elicit delayed dermal hypersensitivity (DTH) responses, they were compared as skin-testing antigens in guinea pigs that were previously sensitized with B . dermatitidis or Histoplasma capsulatum killed whole yeast cells . All 8 of the lots of antigen detected antibody in the sera from dogs with blastomycosis (absorbance values ranged from 0.432 to 0.543; mean value of 0.508) . The absorbance values ranged from 0.283 to 0.439 (mean value of 0.326) when the lysates were assayed against sera from dogs with histoplasmosis . All of the antigens were able to elicit a DTH response in B . dermatitidis immunized animals (mean axes of induration values ranged from 10.5 mm to 12.5 mm; mean value of 11.6 mm) . In contrast, only minimal cross-reactivity was evidenced in the guinea pigs immunized with H . capsulatum (mean axes of induration values ranged from 0 to 4.5 mm; mean value of induration of 1.7 mm). Genetics, 1998 Jan, 148(1), 317 - 29 Regena (Rga), a Drosophila homolog of the global negative transcriptional regulator CDC36 (NOT2) from yeast, modifies gene expression and suppresses position effect variegation; Frolov MV et al.; A mutation in Regena (Rga) was isolated in screens for modifiers of white eye color gene expression . The reduction in the level of the Rga product results in a complex modulation of white mRNA both positively and negatively, depending on the developmental stage . In addition to white, Rga also affects the expression of several other tested genes, with one of them, Vinculin, being regulated in a strong sex-specific manner . Rga was cloned by transposon tagging . Its predicted product lacks any recognized nucleic acid-binding motif but is homologous to a global negative transcriptional regulator, CDC36 (NOT2), from yeast . Rga also acts as a suppressor of position effect variegation, suggesting that a possible function of Rga could be mediation of an interaction between chromatin proteins and the transcriptional complex. Genetics, 1998 Jan, 148(1), 251 - 65 Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2; Elfring LK et al.; The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2 . Here, we report the phenotype of null and dominant-negative brm mutations . Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult . A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine . This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex . Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability . Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism . Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein . Domain II is essential for brm function and is required for the assembly or stability of the BRM complex . In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype. Genetics, 1998 Jan, 148(1), 123 - 30 The Neurospora aab-1 gene encodes a CCAAT binding protein homologous to yeast HAP5; Chen H et al.; The expression of the am (glutamate dehydrogenase) gene is dependent upon two upstream activating sequences, designated URSam(alpha) and URSam(beta) . A heteromeric nuclear protein Am Alpha Binding protein (AAB) binds specifically to a CCAAT box within the URSam(alpha) element . AAB appears to be composed of three components . We used polyclonal antiserum raised against the highly purified AAB1 subunit to isolate a partial aab-1 cDNA clone, which was then used to isolate a full-length cDNA and a genomic clone . The full-length cDNA has the potential to encode a 272 amino acid protein with a calculated molecular weight of 30 kD . Amino acid sequence obtained by Edman analysis of the AAB1 protein confirmed that the aab-1 gene had been cloned . AAB-1 shows similarity to the HAP5 protein of yeast and the CBF-C protein of rat . Each of these proteins is an essential subunit of their respective heteromeric CCAAT binding proteins . The aab1 gene maps on linkage group III of Neurospora crassa near the trp-1 locus . Disruption of the aab-1 gene results in pleiotropic effects on growth and development as well as a 50% reduction in glutamate dehydrogenase levels . Transformation of the aab-1 disruption mutant strain with the cloned genomic copy of the aab-1 gene rescued all of the phenotypic alterations associated with the aab-1 mutation. Biol Reprod, 1998 Feb, 58(2), 302 - 11 Yeast two-hybrid: so many interactions, (in) so little time..; Young KH; Protein-protein interactions are essential to cellular mechanisms at all levels in biologically responsive systems . These interactions occur extracellularly and include ligand-receptor interactions, cell adhesion, antigen recognition, and virus-host recognition . Intracellular protein-protein interactions occur in the formation of multi-protein complexes, during the assembly of cytoskeletal elements, and between receptor-effector, as well as effector-effector, molecules of signal transduction pathways . Finally, assembly of transcriptional machinery involves protein interactions . The yeast two-hybrid method is a powerful technique for analyzing these protein-protein interactions . Since the publication of this technique in the late 1980s, the robust nature and far-reaching utility of yeast two-hybrid systems for functional expression library cloning has led to the identification of many novel proteins in all areas of biological life science research . Additionally, two-hybrid techniques provide a rapid and versatile system for the further characterization of discrete protein-protein interactions . Recent variations on the basic system have enabled application well beyond protein pairs, to investigate multi-protein complexes and protein-nucleotide interactions . Yeast two-hybrid methods necessitate expression and subsequent interaction between a "protein of interest" functional pair within the yeast cell, ultimately driving reporter gene expression and thus effectively linking protein-protein interaction(s) to a change in yeast cell phenotype . Functional protein-protein interactions using the two-hybrid techniques have been demonstrated for all levels of cellular biology; however, until recently, extracellular protein-protein interactions were excluded from investigations using this technique . Investigations from several labs have now demonstrated that extracellular proteins can be studied using two-hybrid methods, thereby enabling intense study of extracellular protein partners using the robust nature and the genetic power of yeast. Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 191 - 8 An N-end rule destabilization mutant reveals pre-Golgi requirements for Sec7p in yeast membrane traffic; Wolf J et al.; Sec7 protein (Sec7p) is required for membrane traffic in the yeast secretory pathway . Because Sec7p regulates more than one stage in the pathway, it has been difficult to assign the most proximal requirement for Sec7p action . We have engineered a novel mutant whose Sec7p levels are regulated by growth conditions and by selective protein destabilization according to the N-end rule . Sec7p depletion causes cell growth arrest and accumulation of transport proteins with post-translational modifications indicative of Sec7p dependence for ER-to-Golgi traffic, in addition to the already characterized Golgi requirements . Immuno-EM of sec7 revealed exaggeration of ER and Golgi membranes with protein accumulation in these exaggerated structures, suggesting that these regions may represent staging areas for cargo sorting and vesicle assembly. Mycoses, 1997 Dec, 40(11-12), 465 - 9 Extracellular enzyme activities of dermatophytes and yeast isolates on solid media; Muhsin TM et al.; A total of 123 isolates of 14 species of dermatophytes and yeasts were screened for the activity of five extracellular enzymes including elastase, keratinase, protease (gelatinase), lipase and phospholipase, by using solid media . The optimal production and activity of each enzyme was determined with regard to different pH and temperatures . Keratinase activity was high with all the tested fungi with exception of Malassezia furfur . Protease (gelatinase) was produced only by dermatophytes . Elastase was secreted by three dermatophytes viz . Microsporum gypseum, Trichophyton mentagrophytes var . mentagrophytes and T . verrucosum, whereas lipase and phospholipase were detected in all the species except T . violaceum. Lipids, 1998 Jan, 33(1), 47 - 58 Stereospecific metabolism of isomeric epoxyoctadecanoic acids in the lactone-producing yeast Sporidiobolus salmonicolor; Haffner T et al.; The metabolic course of four isomeric epoxyfatty acids derived from oleic-, elaidic-, (Z)-, and (E)-vaccenic acids in the lactone-producing yeast, Sporidiobolus salmonicolor, was studied by using the deuterium-labeled precursors . Dihydroxy-, hydroxyoxo-, and hydroxy fatty acids as well as gamma-lactones were identified as metabolic intermediates . Quantitative analysis of the label content and estimation of the enantiomeric composition of the lactones established that, in the first step, the racemic epoxyfatty acids were enantiospecifically hydrolyzed by an epoxide hydrolase . During the subsequent metabolism, the stereochemical orientation of the hydroxy groups of the dihydroxyfatty acids were modified by an oxidation/reduction step. Biochemistry, 1998 Jan 13, 37(2), 615 - 21 Topography of the yeast ATP synthase F0 sector by using cysteine substitution mutants . Cross-linkings between subunits 4, 6, and f; Spannagel C et al.; The arrangement of the N-terminal part of subunit 4 (subunit b) has been studied by the use of mutants containing cysteine residues in a loop connecting the two N-terminal postulated membrane-spanning segments . Labelling of the mutated subunit 4 by the fluorescent probe N-(7-(dimethylamino)-4-methyl-3-coumarinyl)maleimide revealed that the sulfhydryl groups were modified upon incubation of intact mitochondria . In addition, the nonpermeant sulfhydryl reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid prevented the 3-(N-maleimidylpropionyl)biocytin labeling of subunit 4D54C, thus showing a location of this residue in the intermembrane space . Cross-linking experiments revealed the proximity of subunits 4 and f . In addition a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighbor relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space. Genes Dev, 1998 Jan 1, 12(1), 45 - 54 The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain; Myers LC et al.; Mediator was resolved from yeast as a multiprotein complex on the basis of its requirement for transcriptional activation in a fully defined system . Three groups of mediator polypeptides could be distinguished: the products of five SRB genes, identified as suppressors of carboxy-terminal domain (CTD)-truncation mutants; products of four genes identified as global repressors; and six members of a new protein family, termed Med, thought to be primarily responsible for transcriptional activation . Notably absent from the purified mediator were Srbs 8, 9, 10, and 11, as well as members of the SWI/SNF complex . The CTD was required for function of mediator in vitro, in keeping with previous indications of involvement of the CTD in transcriptional activation in vivo . Evidence for human homologs of several mediator proteins, including Med7, points to similar mechanisms in higher cells. Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 253 - 7 Marginal fitness contributions of nonessential genes in yeast; Thatcher JW et al.; Analysis of the complete genome sequence of Saccharomyces cerevisiae confirms and extends earlier evidence that a majority of yeast genes are not essential, at least under laboratory conditions . Many fail to yield a discernible mutant phenotype even when disrupted . Genes not subject to natural selection would accumulate inactivating mutations, so these "cryptic" genes must have functions that are overlooked by the standard methods of yeast genetics . Two explanations seem possible: (i) They have important functions only in environments not yet duplicated in the laboratory and would have conditional phenotypes if tested appropriately . (ii) They make small, but significant, contributions to fitness even under routine growth conditions, but the effects are not large enough to be detected by conventional methods . We have tested the second "marginal benefit" hypothesis by measuring the fitnesses of a random collection of disruption mutants in direct competition with their wild-type progenitor . A substantial majority of mutant strains that lack obvious defects nevertheless are at a significant selective disadvantage just growing on rich medium under normal conditions . This result has important implications for efforts to understand the functions of novel genes revealed by sequencing projects. Curr Biol, 1997 Dec 1, 7(12), 969 - 76 Programmed translational frameshifting in a gene required for yeast telomere replication; Morris DK et al.; BACKGROUND: Telomeres are replicated in most eukaryotes by the enzyme telomerase, a specialized reverse transcriptase . A genetic screen in Saccharomyces cerevisiae designed to detect telomerase components previously led to the identification of four EST ('ever shorter telomeres') genes which are required for telomerase function in vivo . This report describes the cloning and characterization of EST3 . RESULTS: We identified a potential site of +1 ribosomal frameshifting in the EST3 coding sequence and demonstrated that translation both upstream and downstream of this site is required for EST3 function . Mutation of EST3 such that it could not frameshift resulted in a strain with the same phenotype as an est3 null mutant, showing that EST3 frameshifting is required for telomere replication . Immunoblot analysis revealed that two proteins were synthesized from EST3: a truncated protein resulting from translation of only the first open reading frame, as well as the full-length 181 amino-acid Est3 protein resulting from translation through the frameshift site . Only the full-length Est3 protein was required for normal EST3 function . CONCLUSIONS: A programmed translational frameshifting mechanism similar to that used by yeast retrotransposons is employed to produce full-length Est3 protein . This is the first example in yeast of a cellular gene that uses frameshifting to make its protein product, and a potential link is suggested between retrotransposition and the telomerase pathway for telomere maintenance. Curr Biol, 1997 Dec 1, 7(12), 921 - 9 An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast; Epp JA et al.; BACKGROUND: Proteins of the IQGAP family have been identified as candidate effectors for the Rho family of GTPases; however, little is known about their cellular functions . The domain structures of IQGAP family members make them excellent candidates as regulators of the cytoskeleton: their sequences include an actin-binding domain homologous to that found in calponin, IQ motifs for interaction with calmodulin, and a GTPase-binding domain . RESULTS: The genomic sequence of Saccharomyces cerevisiae revealed a single gene encoding an IQGAP family member (denoted IQGAP-related protein: Iqg1) . Iqg1 and IQGAPs share similarity along their entire length, with an amino-terminal calponin-homology (CH) domain, IQ repeats, and a conserved carboxyl terminus . In contrast to IQGAPs, Iqg1 lacks an identifiable GAP motif, a WW domain, and IR repeats, although the functions of these domains in IQGAPs are not well defined . Deletion of the IQG1 gene resulted in lethality . Cellular defects included a deficiency in cytokinesis, altered actin organization, aberrant nuclear segregation, and cell lysis . The primary defect appeared to be a cytokinesis defect, and the other problems possibly arose as a consequence of this initial defect . Consistent with a role in cytokinesis, Iqg1 co-localizes with an actin ring encircling the mother-bud neck late in the cell cycle -a putative cytokinetic ring . IQG1 overexpression resulted in premature actin-ring formation, suggesting that Iqg1 activity temporally controls formation of this structure during the cell cycle . CONCLUSIONS: Yeast IQGAP-related protein, Iqg1, is an important regulator of cellular morphogenesis, inducing actin-ring formation in association with cytokinesis. Curr Opin Genet Dev, 1997 Dec, 7(6), 771 - 6 Functional analysis of the yeast genome; Winzeler EA et al.; The release of the complete genome sequence of the yeast Saccharomyces cerevisiae has ushered in a new phase of genome research in which sequence function will be assigned . The goal is to determine the biological function of each of the > 6,000 open reading frames in the yeast genome . Innovative approaches have been developed that exploit the sequence data and yield information about gene expression levels, protein levels, subcellular localization and gene function for the entire genome. Mycopathologia, 1997, 138(3), 109 - 15 Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii; Aquino-Pinero EE et al.; Protein kinase C (PKC) plays an important role in the control of proliferation and differentiation of a wide range of cell types, and fungi are no exception . Previous results reported by us on the effects of the phorbol ester, 12-myristate-13-acetate phorbol (PMA) and other PKC effector molecules, on dimorphism in Sporothrix schenckii suggested the presence of this enzyme in the fungus and its involvement in the control of morphogenetic transitions . The work summarized here confirms the presence of PKC in yeast and mycelium extracts of S . schenckii . Different isoforms of this enzyme were found to be present in the yeast and mycelium forms of the fungus and were identified by Western blot analysis using affinity purified anti-PKC isoforms specific antibodies: the gamma and zeta isoforms were detected in both the yeast and mycelium forms of the fungus, while the beta isoform was only detected in the yeast form . The presence of PKC was confirmed biochemically by measuring total enzyme activity in both forms of the fungus . No significant differences were observed for the PKC activity level recorded for both the mycelium and yeast forms of the fungus (p < or = 0.05) . These data confirm the presence of PKC activity in Sporothrix schenckii and constitutes the first evidence concerning the differential expression of PKC isoforms in the mycelium and yeast forms of a dimorphic fungus, supporting the possible involvement of this important signal transduction enzyme in the control of morphogenesis in this fungus. Biochim Biophys Acta, 1998 Jan 8, 1379(1), 107 - 17 N-glycosylation does not affect assembly and targeting of proglycinin in yeast; Katsube T et al.; Glycinin, a simple protein, and beta-conglycinin, a glycoprotein, are the dominant storage proteins of soybean and are suggested to be derived from a common ancestor . To investigate why glycinin does not require glycosylation for its maturation, we attempted N-glycosylation of proglycinin A1aB1b using site-directed mutagenesis and yeast expression system . An N-glycosylation consensus sequence Asn-X-Ser/Thr was created at positions 103, 183, 196, 284 and 457 in the variable regions being strongly hydrophilic revealed from the alignment of amino acid sequences of various glycinin-type proteins . Among five mutant proglycinins (Q103N, H183N, G198T, S284N, N459T), Q103N was fully glycosylated, and H183N and N459T were partly (around 20% of the expressed proteins), whereas others were barely or not glycosylated . The glycosylated proglycinin was susceptible to endo-beta-N-acetylglucosamidase and N-glycanase cleavages . N-glycosylation did not cause inconveniences to processing of signal peptide, assembly into trimers and targeting into the vacuoles . Thermal and trypsin sensitivity analyses of the glycosylated proglycinin suggested that N-linked glycan prevents protein-protein interaction but does not stabilize the protein conformation . The reason why glycinin does not require N-glycosylation for its maturation is discussed. Biochim Biophys Acta, 1998 Jan 8, 1379(1), 7 - 15 Effects of cetyltrimethylammonium bromide (CTAB) surfactant upon the dielectric properties of yeast cells; Raicu V et al.; The dielectric properties of yeast cells in the absence and presence of cetyltrimethylammonium bromide (CTAB) were investigated . The surfactant concentration range was between 0.0 and 1.0 mM . The experimental permittivity and conductivity spectra of frequency were analyzed by means of the two-shell electrical cell model (Irimajiri et al., Bull . Inst . Chem . Res., Kyoto Univ . 69 (1991) 421-438), and the electrical phase parameters of cells were subsequently evaluated . The cytoplasm conductivity and the conductivity of the vacuole interior decreased drastically by treating the cells with surfactant . The apparent capacitance of the plasma membrane increased systematically from 0.65 microF/cm2, for untreated cells, up to about 0.75 microF/cm2, at 0.3 mM CTAB . This growth was ascribed to the increase in the folding of the membrane surface associated with the surfactant-induced cell shrinkage . A further addition of the surfactant entailed a gradual decrease of the capacitance that was assigned to the membrane solubilization by the surfactant molecules . Within the accuracy of the data, the specific capacitance of the vacuole membrane was nearly constant (0.544+/-0.021 microF/cm2) over the whole surfactant concentration range . Also, the cytoplasm permittivity remained constant at 64.3+/-4.5. FEBS Lett, 1998 Jan 16, 421(3), 252 - 8 Molecular cloning of human homolog of yeast GAA1 which is required for attachment of glycosylphosphatidylinositols to proteins; Hiroi Y et al.; Anchoring proteins to cell surface membranes by glycosylphosphatidylinositols (GPIs) is important . We have isolated a component of the putative transamidase machinery, hGaa1p (human GPI anchor attachment protein) . hGAA1 cDNA is approximately 2 kb in length and codes 621 amino acids . The amino acid sequence of hGaa1p is 25%, identical and 57% homologous to that of yeast Gaa1p . Moreover, Kite-Dolittle hydrophobicity plots of both proteins show marked similarity . hGAA1 gene is expressed ubiquitously and mRNA levels are higher in the undifferentiated state . Overexpression of antisense hGAA1 in human K562 cells significantly reduced the production of a reporter GPI-anchored protein. J Mol Biol, 1998 Jan 30, 275(4), 547 - 60 Position-specific inhibition of yeast mitochondrial transcription by a poly(T) sequence; Biswas TK et al.; The 3' flanking nucleotide(s) of the octanucleotide promoter sequence regulates transcriptional efficiency of some mitochondrial genes in Saccharomyces cerevisiae . To understand this regulation the in vitro transcriptional activity of various synthetic mitochondrial promoters carrying different 3' flanking sequences was examined . The results presented here demonstrate that consecutive thymidine residues, but no other polynucleotides or secondary structure, in the promoter-proximal non-transcribed DNA strand inhibited mitochondrial transcription . The location and the number of T residues in the cluster as well as the concentration of UTP in the transcription reaction are the important factors determining this transcriptional inhibition . For example, a pair of thymidine nucleotides at positions +2 and +3 is sufficient for inactivation of mitochondrial transcription, whereas more than three consecutive thymidine nucleotides beyond these positions are required for inhibition of mitochondrial transcription . However, a cluster of six to 12 thymidine residues beyond position +11, a point where mtRNA polymerase has been shown to form a stable transcription complex, did not interfere with mitochondrial transcription . Interestingly, at low UTP concentration the mtRNA polymerase generates a large quantity of aborted initiation products on a template carrying promoter-proximal poly(T) sequence probably due to the inability of the polymerase to clear this promoter . On the other hand at high UTP concentration the same mtRNA polymerase on the same mitochondrial promoter produces a higher level of productive initiation complex . These observations suggest that the mechanism of poly(T) inhibition of mitochondrial transcription is a UTP-limited transcriptional attenuation at the promoter site, which might occur under specific physiological conditions (i.e . glucose repression-derepression, switching of aerobic-anaerobic conditions). Biochem Biophys Res Commun, 1998 Jan 26, 242(3), 502 - 7 Identification of the first mammalian sphingosine phosphate lyase gene and its functional expression in yeast; Zhou J et al.; Sphingosine-1-phosphate (S-1-P) has been shown to participate in the proliferative signal transduction pathways of mammalian cells . Sphingosine-1-phosphate lyase (SPL) catalyzes the breakdown of S-1-P . Using the C . elegans SPL nucleotide sequence, we identified a mouse EST as a putative candidate for the homologous gene encoding this enzyme . Sequencing of the mouse EST revealed an open reading frame of 1707 nucleotides . This putative mouse SPL gene is 62% similar and 39% identical to the C . elegans SPL gene and 59% homologous and 39.6% identical to the yeast SPL gene . Expression of the mouse SPL gene in a yeast strain-delta bst1, which carries a deletion of the SPL gene and is hypersensitive to sphingosine, restored a sphingosine-resistant phenotype, suggesting this mouse gene can functionally complement the yeast defect when expressed . In vitro enzyme assay using extracts from these sphingosine-resistant transformants confirmed the SPL activities encoded by this mouse cDNA clone . Northern analysis indicated the mouse SPL gene is expressed at various levels in different tissues . Chromosomal localization mapped this SPL gene to Chromosome 10 at 32 cM . Here, we report the identification of the first mammalian sphingosine phosphate lyase gene. J Dent Assoc S Afr, 1997 Jan, 52(1), 19 - 23 Yeast counts as a measure of host resistance in dental patients; Sykes LM et al.; This study was undertaken to determine whether yeast counts could be used as an indicator of decreased host resistance . One hundred subjects were investigated, 20 young healthy dentate and 20 edentulous adults, 20 adult and 20 geriatric denture wearers and 20 patients fitted with obturators and dentures . Eleven of these obturators were silicone and 9 were acrylic . The denture and the palate of each subject was sampled by taking Sabourauds agar impressions of the posterolateral palatal area and incubating the impressions aerobically at 37 degrees C for 48 hours . The number of yeast colonies were counted and the findings analysed statistically . Palates and dentures of debilitated patients showed high yeast counts and it is concluded that a decreased host resistance can be determined by the method used in this investigation. J Mol Endocrinol, 1997 Dec, 19(3), 321 - 35 Two complementary bioassays for screening the estrogenic potency of xenobiotics: recombinant yeast for trout estrogen receptor and trout hepatocyte cultures; Petit F et al.; A relation between the chemical structure of a xenobiotic and its steroidal action has not yet been clearly established . Thus, it is not possible to define the estrogenic potency of different xenobiotics . An assessment may be accomplished by the use of different bioassays . We have previously developed a yeast system highly and stably expressing rainbow trout estrogen receptor (rtER) in order to analyze the biological activity of the receptor . The recombinant yeast system appears to be a reliable, rapid and sensitive bioassay for the screening and determination of the direct interaction between ER and estrogenic compounds . This system was used in parallel with a more elaborate biological system, trout hepatocyte aggregate cultures, to examine the estrogenic potency of a wide spectrum of chemicals commonly found in the environment . In hepatocyte cultures, the vitellogenin gene whose expression is principally dependent upon estradiol was used as a biomarker . Moreover, competitive binding assays were performed to determine direct interaction between rtER and xenobiotics . In our study, 50% of the 49 chemical compounds tested exhibited estrogenic activity in the two bioassays: the herbicide diclofop-methyl; the fungicides biphenyl, dodemorph, and triadimefon; the insecticides lindane, methyl parathion, chlordecone, dieldrin, and endosulfan; polychlorinated biphenyl mixtures; the plasticizers or detergents alkylphenols and phthalates; and phytoestrogens . To investigate further biphenyl estrogenic activity, its principal metabolites were also tested in both bioassays . Among these estrogenic compounds, 70% were able to activate rtER in yeast and hepatocytes with variable induction levels according to the system . Nevertheless, 30% of these estrogenic compounds exhibited estrogenic activity in only one of the bioassays, suggesting the implication of metabolites or different pathways in the activation of gene transcription . This paper shows that it is important to combine in vivo bioassays with in vitro approaches to elucidate the mechanism of xenoestrogen actions. J Cell Biol, 1998 Jan 12, 140(1), 29 - 37 Prospore membrane formation defines a developmentally regulated branch of the secretory pathway in yeast; Neiman AM; Spore formation in yeast is an unusual form of cell division in which the daughter cells are formed within the mother cell cytoplasm . This division requires the de novo synthesis of a membrane compartment, termed the prospore membrane, which engulfs the daughter nuclei . The effect of mutations in late-acting genes on sporulation was investigated . Mutation of SEC1, SEC4, or SEC8 blocked spore formation, and electron microscopic analysis of the sec4-8 mutant indicated that this inability to produce spores was caused by a failure to form the prospore membrane . The soluble NSF attachment protein 25 (SNAP-25) homologue SEC9, by contrast, was not required for sporulation . The absence of a requirement for SEC9 was shown to be due to the sporulation-specific induction of a second, previously undescribed, SNAP-25 homologue, termed SPO20 . These results define a developmentally regulated branch of the secretory pathway and suggest that spore morphogenesis in yeast proceeds by the targeting and fusion of secretory vesicles to form new plasma membranes in the interior of the mother cell . Consistent with this model, the extracellular proteins Gas1p and Cts1p were localized to an internal compartment in sporulating cells . Spore formation in yeast may be a useful model for understanding secretion-driven cell division events in a variety of plant and animal systems. J Biol Chem, 1998 Jan 9, 273(2), 1247 - 51 Apolipoprotein(a) yeast artificial chromosome transgenic rabbits . Lipoprotein(a) assembly with human and rabbit apolipoprotein B; Rouy D et al.; The in vivo analysis of lipoprotein(a) (Lp(a)), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals . Although transgenic mice have been created containing Lp(a), the relatively small size of the mouse has precluded some studies . To examine the properties of this molecule in a significantly larger mammal, we have used a 270-kilobase yeast artificial chromosome clone containing the human apolipoprotein(a) (apo(a)) gene and a 90-kilobase P1 phagemid clone containing the human apolipoprotein B (apoB) gene to create transgenic rabbits that express either or both transgenes . Expression of both transgenes was tissue specific and localized predominantly to the liver . Average apolipoprotein plasma levels in the rabbits were 2.5 mg/dl for apo(a) and 17.6 mg/dl for human apoB . In contrast to observations in apo(a) transgenic mice, we found that apo(a) plasma levels in the rabbits were stable throughout sexual maturity . Also, apo(a) formed a covalent association with the endogenous rabbit apoB albeit with a lower efficiency than its association with human apoB . The analysis of Lp(a) transgenic rabbits has provided new insights into apo(a) expression and Lp(a) assembly . In addition, these transgenic rabbits potentially will provide an improved experimental model for the in vivo analysis of Lp(a) and its role in promoting atherosclerosis and restenosis. J Biol Chem, 1998 Jan 9, 273(2), 1107 - 13 A debilitating mutation in transcription factor IIE with differential effects on gene expression in yeast; Tijerina P et al.; The influence of transcription factor (TF) IIE on mRNA synthesis in vivo was examined in a temperature-sensitive yeast mutant . A missense mutation in the conserved zinc finger domain severely weakened TFIIE's transcription activity without appreciably affecting its quaternary structure, chromatographic properties, or cellular abundance . The mutation conferred recessive slow-growth and heat-sensitive phenotypes in yeast, but quantitative effects on promoter utilization by RNA polymerase II ranged from strongly negative to somewhat positive . Heat-induced activation of the HSP26, HSP104, and SSA4 genes was attenuated in the mutant, indicating dependence on TFIIE for maximal rates of de novo synthesis . Constitutive HSP expression in mutant cells was elevated, exposing a negative (likely indirect) influence by TFIIE in the absence of heat stress . Our results corroborate and extend recent findings of differential dependence on TFIIE activity for yeast promoters, but reveal an important counterpoint to the notion that dependence is tied to TATA element structure (Sakurai, H., Ohishi, T., and Fukasawa, T . (1997) J . Biol . Chem . 272, 15936-15942) . We also provide empirical evidence for conservation of structure-activity relationships in TFIIE's zinc finger domain, and establish a direct link between TFIIE's biochemical activity in reconstituted transcription and its function in cellular mRNA synthesis. Am J Physiol, 1998 Jan, 274(1 Pt 2), F34 - 42 Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles; Coury LA et al.; Aquaporins 1 (AQP1) and 2 (AQP2) were expressed in the yeast secretory mutant sec6-4 . The mutant accumulates post-Golgi, plasma membrane-targeted vesicles and may be used to produce large quantities of membrane proteins . AQP1 or AQP2 were inducibly expressed in yeast and were localized within isolated sec6-4 vesicles by immunoblot analysis . Secretory vesicles containing AQP1 and AQP2 exhibited high water permeabilities and low activation energies for water flow, indicating expression of functional AQP1 and AQP2 . AQP1 solubilized from secretory vesicles was successfully reconstituted into proteoliposomes, demonstrating the ability to use the yeast system to express aquaporins for reconstitution studies . The AQP2-containing secretory vesicles showed no increased permeability toward formamide, urea, glycerol, or protons compared with control vesicles, demonstrating that AQP2 is highly selective for water over these other substances . We conclude that the expression of aquaporins in yeast sec6 vesicles is a valid system to further study mammalian water channel function. Cell Struct Funct, 1997 Oct, 22(5), 501 - 9 Mutational analysis of Csc1/Vps4p: involvement of endosome in regulation of autophagy in yeast; Shirahama K et al.; In the yeast Saccharomyces cerevisiae, autophagy, a bulk protein degradation in the vacuole, is induced in response to nutrient starvation . In a screen for mutations that result in induction of autophagy even in the presence of nutrients, we have isolated four mutants representing two csc complementation groups . These mutants induce autophagy of which activity is represented by activation of truncated alkaline phosphatase that is designed to be expressed in the cytosol . CSC1 was cloned by complementation of loss of viability phenotype of csc1-1 mutant and shown to be identical to END13/VPS4/GRD13 . Though csc1-1 mutation is recessive, cells of delta csc1 do not induce autophagy in rich media, suggesting that csc1-1 allele is not a complete loss-of-function . Csc1p is a member of novel ATPase family named AAA protein including Sec18p/NSF, Cdc48p/p97, and Pas8p . Mutation site in csc1-1 is found in the SRH region that is highly conserved among AAA proteins . Cells of csc1-1 show sorting defect of CPY and the appearance of the class E compartment . These mutant phenotypes suggest the role of the protein that is involved in the traffic among the Golgi, endosome, and the vacuole in autophagy. Methods, 1997 Oct, 13(2), 190 - 207 Yeast as a model system to study drugs effective against apicomplexan proteins; Sibley CH et al.; Biochemical and genetic analyses are required to identify potential drug targets in apicomplexan parasites, but these studies have proved difficult in most parasite systems . We have developed methods based on expression of parasite proteins in the budding yeast, Saccharomyces cerevisiae, to rapidly screen drugs directed against particular parasite targets, to study the structure and function of these target molecules, and to identify mutations in the parasite genes that alter enzyme specificity or drug sensitivity . In this paper we outline the parameters that need to be considered to design yeast strains that function efficiently to assay function of parasite proteins . Basic protocols and methods are included . We detail some problems that might be encountered in the engineering of these yeast strains and suggest possible solutions . J Biol Chem, 1997 Dec 12, 272(50), 31877 - 84 Yrb2p is a nuclear protein that interacts with Prp20p, a yeast Rcc1 homologue; Taura T et al.; A conserved family of Ran binding proteins (RBPs) has been defined by their ability to bind to the Ran GTPase and the presence of a common region of approximately 100 amino acids (the Ran binding domain) . The yeast Saccharomyces cerevisiae genome predicts only three proteins with canonical Ran binding domains . Mutation of one of these, YRB1, results in defects in transport of macromolecules across the nuclear envelope (Schlenstedt, G., Wong, D . H., Koepp, D . M., and Silver, P . A . (1995) EMBO J . 14, 5367-5378) . The second one, encoded by YRB2, is a 327-amino acid protein with a Ran binding domain at its C terminus and an internal cluster of FXFG and FG repeats conserved in nucleoporins . Yrb2p is located inside the nucleus, and this localization relies on the N terminus . Results of both genetic and biochemical analyses show interactions of Yrb2p with the Ran nucleotide exchanger Prp20p/Rcc1 . Yrb2p binding to Gsp1p (yeast Ran) as well as to a novel 150-kDa GTP-binding protein is also detected . The Ran binding domain of Yrb2p is essential for function and for its association with Prp20p and the GTP-binding proteins . Taken together, we suggest that Yrb2p may play a role in the Ran GTPase cycle distinct from nuclear transport. J Biol Chem, 1997 Dec 12, 272(50), 31829 - 36 Differential ubiquitin-dependent degradation of the yeast apo-cytochrome c isozymes; Pearce DA et al.; The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1- and iso-2-cytochrome c, which are encoded by the nuclear genes CYC1 and CYC7, respectively . The cytochromes c are synthesized in the cytosol, imported into mitochondria, and subsequently modified by the covalent attachment of heme through the action of cytochrome c heme lyase, which is encoded by CYC3 . Apo-iso-2-cytochrome c but not apo-iso-1-cytochrome c was observed in cyc3(-) mutants . Furthermore, pulse-chase experiments previously demonstrated that the lack of apo-iso-1-cytochrome c was due to its rapid degradation . We report herein that this degradation of apo-iso-1-cytochrome c is dependent on ubiquitination and on the action of the proteasome . Diminished degradation of apo-iso-1-cytochrome c was observed in pre2-2 and pre1-1 mutants having altered proteasome subunits; in ubc1, ubc4, and ubc5 strains lacking one or more of the ubiquitin-conjugating enzymes; and in strains blocked in multi-ubiquitination by overproduction of the abnormal ubiquitin-K48R ubiquitin . In addition, we have used epitope-tagged ubiquitin to demonstrate that apo-iso-1-cytochrome c but not apo-iso-2-cytochrome c is ubiquitinated . Furthermore, the degradation of apo-iso-1-cytochrome c was diminished when the N-terminal region was replaced with the N-terminal region of apo-iso-2-cytochrome c, indicating that this region may be the target for degradation . We suggest that ubiquitin-dependent degradation of apo-iso-1-cytochrome c is part of the regulatory process controlling the preferential expression of the iso-cytochromes c. J Biol Chem, 1997 Dec 12, 272(50), 31630 - 5 The type of basal promoter determines the regulated or constitutive mode of transcription in the common control region of the yeast gene pair GCY1/RIO1; Angermayr M et al.; The yeast genes, GCY1 and RIO1, are transcribed divergently from the 869-base pair intergenic region . GCY1 is inducible by galactose about 25-fold due to Gal4p-binding to a single UASGAL, whereas RIO1 is constitutively expressed . GCY1 has a TATA box obeying the consensus TATAAA, whereas the RIO1 5'-upstream region lacks such a motif . In vitro mutagenesis of the TATA motif of GCY1, on the one hand, and introduction of a TATA-element into the promoter of RIO1, on the other hand, as well as inversion of the intergenic region have revealed that transcription of GCY1 and RIO1 is only regulated by Gal4p when a consensus TATA motif is included in their core promoters but not in its absence . The data imply that only transcription complexes that assemble at a consensus TATA box are compatible with specific transactivators, such as Gal4p . As a result, the adjacent gene is subject to regulated expression . By contrast, if a consensus TATA sequence is absent, the initiation complex does not respond to regulatory transcription factors, and consequently, the respective gene is constitutively transcribed . On the other hand, we show that two blocks of homo-oligomeric (dA.dT) sequences do not function as boundary sequences that might confine regulatory action of Gal4p to GCY1. J Biol Chem, 1997 Dec 12, 272(50), 31482 - 8 Heterodimerization-independent functions of cell death regulatory proteins Bax and Bcl-2 in yeast and mammalian cells; Zha H et al.; The pro-apoptotic protein Bax can homodimerize with itself and heterodimerize with the anti-apoptotic protein Bcl-2, but the significance of these protein-protein interactions remains unclear . Alanine substitution mutations were created in a well conserved IGDE motif found within the BH3 domain of Bax (residues 66-69) and the resulting mutant Bax proteins were tested for ability to homodimerize with themselves and to heterodimerize with Bcl-2 . Correlations were made with cell death induction by these mutants of Bax both in mammalian cells where Bax may function through several mechanisms, and in yeast where Bax may exert its lethal actions through a more limited repertoire of mechanisms perhaps related to its ability to form ion channels in intracellular membranes . Two of the mutants, Bax(D68A) and Bax(E69A), retained the ability to homodimerize but failed to interact with Bcl-2 as determined by yeast two-hybrid assays and co-immunoprecipitation analysis using transfected mammalian cells . The Bax(E69A) protein exhibited a lethal phenotype in yeast, which could be specifically suppressed by co-expression of Bcl-2, despite its failure to dimerize with Bcl-2 . Both the Bax(D68A) and Bax(E69A) proteins induced apoptosis when overexpressed in human 293 cells, despite an inability to bind to Bcl-2 . Moreover, co-expression of Bcl-2 with Bax(D68A) and Bax(E69A) rescued mammalian cells from apoptosis . In contrast, a mutant of Bax lacking the IGDE motif, Bax(DeltaIGDE), was incapable of either homodimerizing with itself or heterodimerizing with Bcl-2 and was inactive at promoting cell death in either yeast or mammalian cells . Although failing to interact with Bcl-2, the Bax(D68A) and Bax(E69A) mutants retained the ability to bind to Bid, a putative Bax-activating member of the Bcl-2 family, and collaborated with Bid in inducing apoptosis . When taken together with previous observations, these findings indicate that (i) Bax can induce apoptosis in mammalian cells irrespective of heterodimerization with Bcl-2 and (ii) Bcl-2 can rescue both mammalian cells and yeast from the lethal effects of Bax without heterodimerizing with it . However, these results do not exclude the possibility that BH3-dependent homodimeriza