Yeasts

Can J Microbiol, 2000 Mar, 46(3), 195 - 210
Ribosomal DNA, species structure, and biogeography of the cactophilic yeast Clavispora opuntiae; Lachance MA et al.; The ribosomal DNA of the cactophilic yeast species Clavispora opuntiae was studied in order to clarify the global distribution of the yeast . Over 500 strains, including isolates from several new localities worldwide, were characterized by rDNA restriction mapping . An unusual restriction pattern previously encountered only in one strain, from Conception Island in the Bahamas, was found in several Brazilian isolates . Sequences of the D1/D2 and D7/D8 divergent domains of the large subunit (LSU) and of the intergenic spacers (IGS) confirmed that these strains represent a genetically distinct variety of Clavispora opuntiae . This divergence had previously been hypothesized on the basis of reduced genetic recombination in inter-varietal crosses and the presence of a polymorphic ApaI restriction site located in the LSU . The exact position of the ApaI site in the D8 divergent domain and the nature of the variation that it reveals were determined . The complete sequences of 12 intergenic spacers clarified the significance of the species-wide variation uncovered by restriction mapping . Most of the polymorphic sites occur in the IGS1 and IGS2 regions, on either side of the 5S gene, and the variation is largely due to differences in the numbers and the sequences of internal repeats . Two other polymorphic sites are located in the external transcribed spacer (ETS) region . The reliability of various sites as indicators of overall spacer sequence divergence differed from one case to another . Variety-specific probes were devised and used to screen 120 strains for the presence of recombinant rDNA spacers . Three strains gave ambiguous results, but these did not constitute evidence that inter-varietal recombination has taken place in nature . The hypothesis that the global movement of Clavispora opuntiae has been influenced by the worldwide biological control of prickly pear with Cactoblastis cactorum, a moth of Argentinian origin, has received additional support from the demonstration that Argentinian strains have rDNAs similar to those found where the moth has been introduced . A dramatic founder effect was identified in a yeast population collected in cacti (Maui, Hawaii) in a site where the moth had been recently introduced.

J Biol Chem, 2000 May 26, 275(21), 15895 - 904
Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange; Song B et al.; Rad51-catalyzed DNA strand exchange is greatly enhanced by the single-stranded (ss) DNA binding factor RPA if the latter is introduced after Rad51 has already nucleated onto the initiating ssDNA substrate . Paradoxically, co-addition of RPA with Rad51 to the ssDNA to mimic the in vivo situation diminishes the level of strand exchange, revealing competition between RPA and Rad51 for binding sites on ssDNA . Rad52 promotes strand exchange but only when there is a need for Rad51 to compete with RPA for loading onto ssDNA . Rad52 is multimeric, binds ssDNA, and targets Rad51 to ssDNA . Maximal restoration of pairing and strand exchange requires amounts of Rad52 substoichiometric to Rad51 and involves a stable, equimolar complex between Rad51 and Rad52 . The Rad51-Rad52 complex efficiently utilizes a ssDNA template saturated with RPA for homologous pairing but does not appear to be more active than Rad51 when an RPA-free ssDNA template is used . Rad52 does not substitute for RPA in the pairing and strand exchange reaction nor does it lower the dependence of the reaction on Rad51 or RPA.

J Biol Chem, 2000 Jun 9, 275(23), 17249 - 55
The transcriptional response of yeast to saline stress; Posas F et al.; Adaptation to changes in extracellular salinity is a critical event for cell survival . Genome-wide DNA chip analysis has been used to analyze the transcriptional response of yeast cells to saline stress . About 7% of the genes encoded in the yeast genome are induced more than 5-fold after a mild and brief saline shock (0.4 m NaCl, 10 min) . Interestingly, most responsive genes showed a very transient expression pattern, as mRNA levels dramatically declined after 20 min in the presence of stress . A quite similar set of genes increased expression in cells subjected to higher saline concentrations (0.8 m NaCl), although in this case the response was delayed . Therefore, our data show that cells respond to saline stress by inducing the expression of a very large number of genes and suggest that stress adaptation requires regulation of many cellular aspects . The transcriptional induction of most genes that are strongly responsive to salt stress was highly or fully dependent on the presence of the stress-activated mitogen-activated protein kinase Hog1, indicating that the Hog1-mediated signaling pathway plays a key role in global gene regulation under saline stress conditions.

J Biol Chem, 2000 Jun 9, 275(23), 17718 - 27
Distinct protein domains of the yeast Golgi GDP-mannose transporter mediate oligomer assembly and export from the endoplasmic reticulum; Gao XD et al.; The substrates for glycan synthesis in the lumen of the Golgi are nucleotide sugars that must be transported from the cytosol by specific membrane-bound transporters . The principal nucleotide sugar used for glycosylation in the Golgi of the yeast Saccharomyces cerevisiae is GDP-mannose, whose lumenal transport is mediated by the VRG4 gene product . As the sole provider of lumenal mannose, the Vrg4 protein functions as a key regulator of glycosylation in the yeast Golgi . We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi . Here, we analyzed epitope-tagged alleles of VRG4 . Gel filtration chromatography and co-immunoprecipitation experiments demonstrate that the Vrg4 protein forms homodimers with specificity and high affinity . Deletion analyses identified two regions essential for Vrg4p function . Mutant Vrg4 proteins lacking the predicted C-terminal membrane-spanning domain fail to assemble into oligomers (Abe, M., Hashimoto, H., and Yoda, K . (1999) FEBS Lett . 458, 309-312) and are unstable, while proteins lacking the N-terminal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmic reticulum (ER) . Fusion of the N terminus of Vrg4p to related ER membrane proteins promote their transport to the Golgi, suggesting that sequences in the N terminus supply information for ER export . The dominant negative phenotype resulting from overexpression of truncated Vrg4-DeltaN proteins provides strong genetic evidence for homodimer formation in vivo . These studies are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positive sorting rather than passive default.

J Biol Chem, 2000 Jun 9, 275(23), 17762 - 70
A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin; Maudoux O et al.; The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified . Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2 . This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants . When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form . Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region . The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated . In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin . Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase . These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity . Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth . We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.

J Biol Chem, 2000 Jun 2, 275(22), 16963 - 8
Limited proteolysis of yeast elongation factor 3 . Sequence and location of the subdomains; Kambampati R et al.; Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi . We used limited proteolysis to study the organization of the subdomains of EF-3 . Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa . Yeast ribosomes protected EF-3 and the large fragments from further degradation . ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments . The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding . Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3 . Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA . Neither tRNA nor rRNA altered the tryptic cleavage pattern . The proteolytic products were sequenced by mass spectrometric analysis . EF-3 is blocked NH(2)-terminally by an acetylated serine . The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin . The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function . The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain . The primary ribosome binding site is located near the loosely structured carboxyl-terminal end.

J Biol Chem, 2000 Jun 9, 275(23), 17754 - 61
Analysis of the role of the hypervariable region of yeast Ras2p and its farnesylation in the interaction with exchange factors and adenylyl cyclase; Crechet JB et al.; Ras proteins from Saccharomyces cerevisiae differ from mammalian Ha-Ras in their extended C-terminal hypervariable region . We have analyzed the function of this region and the effect of its farnesylation with respect to the action of the GDP/GTP exchange factors (GEFs) Cdc25p and Sdc25p and the target adenylyl cyclase . Whereas Ras2p farnesylation had no effect on the interaction with purified GEFs from the Cdc25 family, this modification became a strict requirement for stimulation of the nucleotide exchange on Ras using reconstituted cell-free systems with GEFs bound to the cell membrane . Determination of GEF effects showed that in cell membrane the Cdc25p dependent activity on Ras2p was predominant over that of Sdc25p . In contrast to full-length GEFs, a membrane-bound C-terminal region containing the catalytic domain of Cdc25p was still able to react productively with unfarnesylated Ras2p . These results indicate that in membrane-bound full-length GEF the N-terminal moiety regulates the interaction between catalytic domain and farnesylated Ras2p.GDP . Differently from GEF, full activation of adenylyl cyclase did not require farnesylation of Ras2p.GTP, even if this step of maturation was found to facilitate the interaction . The use of Ha-Ras/Ras2p chimaeras of different length emphasized the key role of the hypervariable region of Ras2p in inducing maximum activation of adenylyl cyclase and for a productive interaction with membrane-bound GEF.

J Biol Chem, 2000 May 26, 275(21), 15709 - 16
Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase; Petrov VV et al.; As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H(+)-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His) . To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles . Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport . Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport . Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled . At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis . Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases . Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.

J Biol Chem, 2000 May 5, 275(18), 13171 - 4
Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast; Osmulski PA et al.; The proteasome is a major cytosolic proteolytic complex, indispensable in eukaryotic cells . The barrel-shaped core of this enzyme, the 20 S proteasome, is built from 28 subunits forming four stacked rings . The two inner beta-rings harbor active centers, whereas the two outer alpha-rings play a structural role . Crystal structure of the yeast 20 S particle showed that the entrance to the central channel was sealed . Because of this result, the path of substrates into the catalytic chamber has remained enigmatic . We have used tapping mode atomic force microscopy (AFM) in liquid to address the dynamic aspects of the 20 S proteasomes from fission yeast . We present here evidence that, when observed with AFM, the proteasome particles in top view position have either open or closed entrance to the central channel . The preferred conformation depends on the ligands present . Apparently, the addition of a substrate to the uninhibited proteasome shifts the equilibrium toward the open conformation . These results shed new light on the possible path of the substrate into the proteolytic chamber.

J Biol Chem, 2000 May 26, 275(21), 15609 - 12
A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast; Oelkers P et al.; The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol . To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases . Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol . By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production . In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor . The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase . These mechanisms may also exist in mammalian cells.

Biochemistry, 2000 Apr 11, 39(14), 4199 - 205
Environmental study of subunit i, a F(o) component of the yeast ATP synthase; Paumard P et al.; The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was determined by the use of cysteine-substituted mutants . The N(in)-C(out) orientation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid . Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl oxidation or reaction with homobifunctional and heterobifunctional reagents . Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit i per complex . Cross-linked products between mutant subunits i and proteins loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at the periphery of the enzyme and interacts with proteins of the inner mitochondrial membrane that are not involved in the structure of the yeast ATP synthase.

Biochemistry, 2000 Apr 11, 39(14), 4082 - 9
Yeast resolving enzyme CCE1 makes sequential cleavages in DNA junctions within the lifetime of the complex; Fogg JM et al.; CCE1 is a DNA junction-resolving enzyme of Saccharomyces cerevisiae . Such enzymes are required to make two symmetrically paired cleavages in order to resolve the four-way junction productively . Using a cruciform assay, we show here that CCE1 introduces two unilateral cleavages in a sequential manner . This requires that the protein remains bound to the junction, preventing branch migration of the point of strand exchange . From a detailed kinetic analysis, we find that the CCE1 cleavage at a given site is accelerated by a factor of 5-10 when it occurs subsequently to the initial cleavage . These properties ensure a productive resolution of the four-way junction and may be general for junction-resolving enzymes.

Biochemistry, 2000 Apr 11, 39(14), 3943 - 54
Characterization of the DNA-binding domains from the yeast cell-cycle transcription factors Mbp1 and Swi4; Taylor IA et al.; The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods . An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity . Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding . Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced . The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity {K(A) approximately (1-4) x 10(6) M(-)(1)} and with a 1:1 stoichiometry . However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide . Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements . Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding . These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity . Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif . Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins.

Genetics, 2000 Apr, 154(4), 1561 - 76
A yeast taf17 mutant requires the Swi6 transcriptional activator for viability and shows defects in cell cycle-regulated transcription; Macpherson N et al.; In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle . Although SBF is required for cell viability, SWI6 is not an essential gene . We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10 . We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Delta and slm8-1 swi6Delta double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6 . Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes . Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex . Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region . The cell cycle and transcriptional defects caused by taf17(slm7-1) are consistent with the role of TAF(II)s as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF.

Genetics, 2000 Apr, 154(4), 1485 - 95
Regulation of the yeast INO1 gene . The products of the INO2, INO4 and OPI1 regulatory genes are not required for repression in response to inositol; Graves JA et al.; The ino2Delta, ino4Delta, opi1Delta, and sin3Delta mutations all affect expression of INO1, a structural gene for inositol-1-phosphate synthase . These same mutations affect other genes of phospholipid biosynthesis that, like INO1, contain the repeated element UAS(INO) (consensus 5' CATGTGAAAT 3') . In this study, we evaluated the effects of these four mutations, singly and in all possible combinations, on growth and expression of INO1 . All strains carrying an ino2Delta or ino4Delta mutation, or both, failed to grow in medium lacking inositol . However, when grown in liquid culture in medium containing limiting amounts of inositol, the opi1Delta ino4Delta strain exhibited a level of INO1 expression comparable to, or higher than, the wild-type strain growing under the same conditions . Furthermore, INO1 expression in the opi1Delta ino4Delta strain was repressed in cells grown in medium fully supplemented with both inositol and choline . Similar results were obtained using the opi1Delta ino2Delta ino4Delta strain . Regulation of INO1 was also observed in the absence of the SIN3 gene product . Therefore, while Opi1p, Sin3p, and the Ino2p/Ino4p complex all affect the overall level of INO1 expression in an antagonistic manner, they do not appear to be responsible for transmitting the signal that leads to repression of INO1 in response to inositol . Various models for Opi1p function were tested and no evidence for binding of Opi1p to UAS(INO), or to Ino2p or Ino4p, was obtained.

Genetics, 2000 Apr, 154(4), 1463 - 71
The fission yeast git5 gene encodes a Gbeta subunit required for glucose-triggered adenylate cyclase activation; Landry S et al.; Fission yeast adenylate cyclase is activated by the gpa2 Galpha subunit of a heterotrimeric guanine-nucleotide binding protein (G protein) . We show that the git5 gene, also required for this activation, encodes a Gbeta subunit . In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Galpha involved in the pheromone response pathway . While 43% identical to mammalian Gbeta's, the git5 protein lacks the amino-terminal coiled-coil found in other Gbeta subunits, yet the gene possesses some of the coding capacity for this structure 5' to its ORF . Although both gpa2 (Galpha) and git5 (Gbeta) are required for adenylate cyclase activation, only gpa2 is needed to maintain basal cAMP levels . Strains bearing a git5 disruption are derepressed for fbp1 transcription and sexual development even while growing in a glucose-rich environment, although fbp1 derepression is half that observed in gpa2 deletion strains . Multicopy gpa2 partially suppresses the loss of git5, while the converse is not true . These data suggest that Gbeta is required for activation of adenylate cyclase either by promoting the activation of Galpha or by independently activating adenylate cyclase subsequent to Galpha stimulation as seen in type II mammalian adenylate cyclase activation.

EMBO J, 2000 Apr 3, 19(7), 1650 - 60
U snRNP assembly in yeast involves the La protein; Xue D et al.; In all eukaryotic nuclei, the La autoantigen binds nascent RNA polymerase III transcripts, stabilizing these RNAs against exonucleases . Here we report that the La protein also functions in the assembly of certain RNA polymerase II-transcribed RNAs into RNPs . A mutation in a core protein of the spliceosomal snRNPs, Smd1p, causes yeast cells to require the La protein Lhp1p for growth at low temperatures . Precursors to U1, U2, U4 and U5 RNAs are bound by Lhp1p in both wild-type and mutant cells . At the permissive temperature, smd1-1 cells contain higher levels of stable U1 and U5 snRNPs when Lhp1p is present . At low temperatures, Lhp1p becomes essential for the accumulation of U4/U6 snRNPs and for cell viability . When U4 RNA is added to extracts, the pre-U4 RNA, but not the mature RNA, is bound by Smd1p . These results suggest that, by stabilizing a 3'-extended form of U4 RNA, Lhp1p facilitates efficient Sm protein binding, thus assisting formation of the U4/U6 snRNP.

Bioinformatics, 1999 Dec, 15(12), 980 - 6
Segmentation of yeast DNA using hidden Markov models; Peshkin L et al.; MOTIVATION: Compositionally homogeneous segments of genomic DNA often correspond to meaningful biological units . Simple sliding window analysis is usually insufficient for compositional segmentation of natural sequences . Hidden Markov models (HMM) with a small number of states are a natural language for description of compositional properties of chromosome-size DNA sequences . RESULTS: The algorithms were applied to yeast Saccharomyces cerevisiae chromosomes (YC) I, III, IV, VI and IX . The optimal number of HMM states is found to be four . The optimal four-state HMMs for all chromosomes are very similar, as well as the reconstructed segmentations . In most cases the models with k + 1 states are obtained by 'splitting' one of the states in the model with k states, and the corresponding increase of the level of detail in segmentation . The high AT states usually correspond to intergenic regions . We also explore the model's likelihood landscape and analyze the dynamics of the optimization process, thus addressing the problem of reliability of the obtained optima and efficiency of the algorithms.

Curr Biol, 2000 Mar 23, 10(6), 329 - 32
Phosphorylation and spindle pole body localization of the Cdc15p mitotic regulatory protein kinase in budding yeast; Xu S et al.; Cdc15p is an essential protein kinase and functions with a group of late mitotic proteins that includes Lte1p, Tem1p, Cdc14p and Dbf2p/Dbf20p to inactivate Cdc28p-Clb2p at the end of mitosis in budding yeast {1} {2} . Cdc14p is activated and released from the nucleolus at late anaphase/telophase to dephosphorylate important regulators of Cdc28p-Clb2p such as Hct1p/Cdh1p, Sic1p and Swi5p in a CDC15-dependent manner {3} {4} {5} {6} {7} . How Cdc15p itself is regulated is not known . Here, we report that both the phosphorylation and localization of Cdc15p are cell cycle regulated . The extent of phosphorylation of Cdc15p gradually increases during cell-cycle progression until some point during late anaphase/telophase when it is rapidly dephosphorylated . We provide evidence suggesting that Cdc14p is the phosphatase responsible for the dephosphorylation of Cdc15p . Using a Cdc15p fusion protein coupled at its carboxyl terminus to green fluorescent protein (GFP), we found that Cdc15p, like its homologue Cdc7p {8} in fission yeast, localizes to the spindle pole bodies (SPBs) during mitosis . At the end of telophase, a portion of Cdc15p is located at the mother-bud neck, suggesting a possible role for Cdc15p in cytokinesis.

J Biol Chem, 2000 Apr 7, 275(14), 10655 - 60
Impaired proteasome function rescues thermosensitivity of yeast cells lacking the coatomer subunit epsilon-COP; Kimata Y et al.; Formation of COPI-coated transport vesicles requires a cytosolic protein complex consisting of seven subunits: alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP, collectively designated coatomer . The yeast Saccharomyces cerevisiae gene encoding the epsilon-COP subunit is known as SEC28/ANU2 . anu2 null mutant cells (anu2Delta) are temperature-sensitive, and alpha-COP is rapidly degraded in these cells when they are shifted to the restrictive temperature . We isolated extragenic suppressors that rescue the temperature-sensitive growth defect of anu2Delta cells . Genetic analysis revealed that one of the suppressors is allelic to PRE8 (PRS4), which encodes a 20 S proteasome subunit . In the presence of a proteasome inhibitor, MG132, anu2Delta cells did not cease growth even at the restrictive temperature . Furthermore, MG132 inhibited the rapid decrease of alpha-COP levels in anu2Delta cells shifted to the restrictive temperature . However, secretion of certain proteins by these cells was impaired even in the presence of MG132 . In conclusion, impairment of proteasome-dependent proteolysis rescued some, but not all, temperature-sensitive defects of anu2Delta cells . These results are discussed in terms of evidence that epsilon-COP plays a critical role in maintaining the structural integrity of alpha-COP.

RNA, 2000 Mar, 6(3), 449 - 57
The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo; Brown JT et al.; The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A . XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA . Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats . An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function . Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex . With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex . Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p . However, the Ski3p/Ski8p interaction did not require Ski2p . In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation . The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8 . Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic . These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.

RNA, 2000 Mar, 6(3), 352 - 68
Splicing enhancement in the yeast rp51b intron; Libri D et al.; Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein-protein interactions . It has been previously shown that, in yeast, communication mediated by RNA-RNA interactions between the two ends of introns is a basis for splicing enhancement . We designed experiments of randomization-selection to isolate splicing enhancers that would work independently from RNA secondary structures . Surprisingly, one of the two families of sequences selected was essentially composed of 5' splice site variants . We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects . The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation . Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement.

Prog Cell Cycle Res, 2000, 4, 97 - 106
Functions of Pho85 cyclin-dependent kinases in budding yeast; Moffat J et al.; Pho85 is a multifunctional cyclin-dependent kinase (Cdk) in Saccharomyces cerevisiae that has emerged as an important model for the role of Cdks in both cell cycle control and other processes . Pho85 was originally discovered as a regulator of phosphate metabolism but roles for Pho85 in glycogen biosynthesis, actin regulation and cell cycle progression have since been discovered . Ten genes encoding known or putative Pho85 cyclins (Pcls) have been identified and the Pcls appear to target Pho85 to specific cellular functions and substrates . In this chapter, we review the functions of the various Pcl-Pho85 complexes in budding yeast . We focus on the known biological roles of Pho85 with an emphasis on Pho85 substrates and cyclin-Cdk specificity.

Prog Cell Cycle Res, 2000, 4, 61 - 9
A Cdc7p-Dbf4p protein kinase activity is conserved from yeast to humans; Johnston LH et al.; DBF4 and CDC7 were identified as budding yeast cell cycle mutants that arrest immediately before S phase . The Dbf4p and Cdc7p proteins interact to form a protein kinase, Cdc7p being the catalytic subunit and Dbf4p is a cyclin-like molecule that activates the kinase in late G1 . Dbf4p also targets Cdc7p to origins of replication where likely substrates include the Mcm proteins . Dbf4p and Cdc7p related proteins occur in the fission yeast and in metazoans . These also phosphorylate Mcm proteins and preliminary evidence indicates a similar function to Dbf4p/Cdc7p in budding yeast . The Dbf4p/Cdc7p activity will therefore very likely be conserved in all eukaryotes.

Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 492 - 4
Crystallization and preliminary X-ray crystallographic analysis of yeast arginyl-tRNA synthetase-yeast tRNAArg complexes; Delagoutte B et al.; Three different crystal forms of complexes between arginyl-tRNA synthetase from the yeast Saccharomyces cerevisae (yArgRS) and the yeast second major tRNA(Arg) (tRNA(Arg)(ICG)) isoacceptor have been crystallized by the hanging-drop vapour-diffusion method in the presence of ammonium sulfate . Crystal form II, which diffracts beyond 2.2 A resolution at the European Synchrotron Radiation Facility ID14-4 beamline, belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 129.64, b = 107.47, c = 71 . 38 A . This crystal form presents the highest resolution obtained for an active form of an aminoacyl-tRNA synthetase-tRNA complex . The estimated V(m) of 2.6 A(3) Da(-1) indicates one molecule of complex in the asymmetric unit . The three crystal forms were solved by the molecular-replacement method using the coordinates of the free yArgRS.

Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 421 - 30
MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites; Erskine PT et al.; MAD experiments attempting to solve the structure of 5--aminolaevulinic acid dehydratase using Zn and Pb edges are described . The data obtained proved insufficient for a complete structure solution but were invaluable in subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved . These sites include the highly inhibitory substitution of an enzymic cofactor Zn(2+) ion by Pb(2+) ions, which represents a major contribution towards understanding the molecular basis of lead poisoning . The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg co-crystal to provide the first delineation of the enzyme's quaternary structure . In this MADIR analysis, the Hg co-crystal data were treated as native data . Anomalous difference Fouriers were again used, revealing that Hg(2+) had substituted for the same Zn(2+) cofactor ion as had Pb(2+), a finding of fundamental importance for the understanding of mercury poisoning . In addition, Pt(2+) ions were found to bind at the same place in the structure . The refined structures of the Pb- and the Hg-complexed enzymes are presented at 2.5 and 3.0 A resolution, respectively.

Proteins, 2000 May 15, 39(3), 204 - 11
Conversion of yeast phosphoglycerate kinase into amyloid-like structure; Damaschun G et al.; Yeast phosphoglycerate kinase is a structurally well-characterized enzyme consisting of 415 amino acids without disulfide bonds . Anion-induced refolding from its acid-unfolded state gives rise to the formation of worm-like amyloid fibrils with a persistence length of 73 nm . Electron microscopy and small-angle X-ray scattering data indicate that the fibrils have an elliptical cross-section with dimensions of 10.2 nm x 5.1 nm . About half of all amino acids are organized in form of cross-beta structure which gives rise to typical infrared spectra, X-ray diffraction and yellow-green birefringence after Congo red staining . The kinetics of amyloid formation, monitored by infrared spectroscopy, dynamic light scattering and X-ray scattering, was found to be strongly dependent on protein concentration . The infrared data indicate that the formation of cross-beta structure practically comes to an end already after some hours, whereas the length-growth of the amyloid fibrils, monitored by small-angle X-ray scattering, was not yet completed after 1,300 hours.

J Biochem Biophys Methods, 2000 Mar 16, 42(3), 133 - 5
A simple approach for the simultaneous isolation and immobilization of invertase using crude extracts of yeast and Jack bean meal; Melo JS et al.; Crude cell-free extract of yeast cells was mixed with sufficient amount of Jack bean meal extract so as to precipitate all the invertase . The precipitate was then cross-linked using 2% glutaraldehyde retaining over 60% of the activity . The immobilized invertase could be reused for over ten batches without loss in activity.

Biochemistry, 2000 Apr 4, 39(13), 3699 - 707
Kinetic analysis of oxygen utilization during cofactor biogenesis in a copper-containing amine oxidase from yeast; Schwartz B et al.; A detailed kinetic analysis of oxygen consumption during TPQ biogenesis has been carried out on a yeast copper amine oxidase . O(2) is consumed in a single, exponential phase, the rate of which responds linearly to dissolved oxygen concentration . This behavior is observed up to conditions of maximally obtainable oxygen concentrations . In contrast, no viscosity effect is observed on rate, implicating a high K(m) for O(2) . Binding of oxygen appears to occur faster than its consumption and to result in displacement of the precursor tyrosine onto copper to form a charge-transfer species, described in the the preceding paper of this issue {Dove, J . E., Schwartz, B., Williams, N . K., and Klinman, J . P . (2000) Biochemistry 39, 3690-3698) . Reaction between this intermediate and O(2) is proposed to occur in a rate-limiting step, and to proceed more rapidly when the tyrosine is deprotonated . This rate-limiting step in cofactor biogenesis does not display a solvent isotope effect and is, thus, uncoupled from proton transfer . Comparisons are drawn between the proposed biogenesis mechanism and that for the oxidation of reduced cofactor during catalytic turnover in the mature enzyme.

Biochemistry, 2000 Apr 4, 39(13), 3611 - 23
X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery; Hall LT et al.; Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD . Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD . The 1.55 A crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods . The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion . In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested . In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions . This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion . Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2 . The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD . The advantages of the dimer-dimer model over the heterodimer model are enumerated.

Nucleic Acids Res, 2000 Apr 15, 28(8), 1700 - 6
Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast; Davis CA et al.; Correct identification of all introns is necessary to discern the protein-coding potential of a eukaryotic genome . The existence of most of the spliceosomal introns predicted in the genome of Saccharomyces cerevisiae remains unsupported by molecular evidence . We tested the intron predictions for 87 introns predicted to be present in non-ribosomal protein genes, more than a third of all known or suspected introns in the yeast genome . Evidence supporting 61 of these predictions was obtained, 20 predicted intron sequences were not spliced and six predictions identified an intron-containing region but failed to specify the correct splice sites, yielding a successful prediction rate of <80% . Alternative splicing has not been previously described for this organism, and we identified two genes (YKL186C/ MTR2 and YML034W) which encode alternatively spliced mRNAs; YKL186C/ MTR2 produces at least five different spliced mRNAs . One gene (YGR225W/ SPO70 ) has an intron whose removal is activated during meiosis under control of the MER1 gene . We found eight new introns, suggesting that numerous introns still remain to be discovered . The results show that correct prediction of introns remains a significant barrier to understanding the structure, function and coding capacity of eukaryotic genomes, even in a supposedly simple system like yeast.

Mol Cell Biol, 2000 Apr, 20(8), 2827 - 38
The role of nuclear cap binding protein Cbc1p of yeast in mRNA termination and degradation; Das B et al.; The cyc1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs which differ in length at their 3' termini . cyc1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex . The upf1-Delta deletion suppressed the cyc1-512 defect by diminishing degradation of the longer subset of cyc1-512 mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations . On the other hand, suppression of cyc1-512 defects by cbc1-Delta occurred by two different mechanisms . The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Delta mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longer cyc1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation . Furthermore, cbc1-Delta greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export . We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.

Bioorg Med Chem, 2000 Mar, 8(3), 571 - 80
Efficient syntheses, human and yeast farnesyl-protein transferase inhibitory activities of chaetomellic acids and analogues; Singh SB et al.; Chaetomellic acids are a class of alkyl dicarboxylic acids that were isolated from Chaetomella acutiseta . They are potent and highly specific farnesyl-pyrophosphate (FPP) mimic inhibitors of Ras farnesyl-protein transferase . We have previously described the first biogenetic type aldol condensation-based total synthesis of chaetomellic acid A . Modification of the later steps of that synthesis resulted in the efficient syntheses of chaetomellic acids A and B in three steps with 75-80% overall yield . In this report, details of the original total syntheses of chaetomellic acids A, B and C, the new syntheses of acids A and B and structure-activity relationship of these compounds against various prenyl transferases including human and yeast FPTase and bovine and yeast GGPTase I are described . Chaetomellic acids are differentially active against human and yeast FPTase . Chaetomellic acid A inhibited human and yeast FPTase activity with IC50 values of 55 nM and 225 microM, respectively . In contrast, chaetomellic acid C showed only a 10-fold differential in inhibitory activities against human versus yeast enzymes . In keeping with molecular modeling-based predictions, the compounds with shorter alkyl side chains (C-8) were completely inactive against FPTase.

Mol Gen Genet, 2000 Feb, 263(1), 90 - 5
Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from Bactrocera oleae by functional complementation in yeast; Benos P et al.; The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution . Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments . However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies . We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants . Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently . Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae . We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes.

Mol Gen Genet, 2000 Feb, 263(1), 81 - 9
Involvement of the inverted repeat of the yeast 2-micron plasmid in Flp site-specific and RAD52-dependent homologous recombination; Storici F et al.; Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs) . However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known . In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp . We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination . In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52 . The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present . Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination.

Mol Gen Genet, 2000 Feb, 263(1), 48 - 59
Activation of basal transcription by a mutation in SIN4, a yeast global repressor, occurs through a mechanism different from activator-mediated transcriptional enhancement; Mizuno T et al.; The Saccharomyces cerevisiae protein Sin4 has been suggested to affect the transcription of various genes by locally altering chromatin structure . Previous studies have defined two classes of promoters: those which are activated by loss of SIN4 function (termed sin4-responsive promoters) and those which are not activated by sin4 mutations (termed sin4 non-responsive promoters) . We analyzed the mechanism of this differential response of the two classes of promoters to a sin4 mutation . The sin4 non-responsive promoters were activated when upstream elements in the promoter region were eliminated . The upstream elements of sin4 non-responsive promoters were, in turn, found to repress the activity of the sin4-responsive promoters in an orientation-independent manner . The sin4-mediated activation was repressed by the Rme1- but not by the Tup1-Ssn6-mediated repression system . Activation of sin4-responsive promoters by Pho4 and the sin4 mutation was additive, and enhancement of transcription driven by sin4-responsive promoters was found to be due to an increase in the basal rate of transcription . The upstream regions in the sin4 non-responsive promoters contained elements that were able to inhibit activation of basal transcription . Based on these observations, we suggest that activation of basal transcription by a mutation in a gene for a global repressor, SIN4, occurs through a mechanism that differs from that responsible for activator-mediated transcriptional enhancement, and we therefore propose that basal transcription and activator-mediated transcription are repressed by different mechanisms.

Science, 2000 Mar 24, 287(5461), 2257 - 9
Molecular linkage underlying microtubule orientation toward cortical sites in yeast; Korinek WS et al.; Selective microtubule orientation toward spatially defined cortical sites is critical to polarized cellular processes as diverse as axon outgrowth and T cell cytotoxicity . In yeast, oriented cytoplasmic microtubules align the mitotic spindle between mother and bud . The cortical marker protein Kar9 localizes to the bud tip and is required for the orientation of microtubules toward this region . Here, we show that Kar9 directs microtubule orientation by acting through Bim1, a conserved microtubule-binding protein . Bim1 homolog EB1 was originally identified through its interaction with adenomatous polyposis coli (APC) tumor suppressor, raising the possibility that an APC-EB1 linkage orients microtubules in higher cells.

Genomics, 2000 Mar 1, 64(2), 211 - 5
CFTR intron 1 increases luciferase expression driven by CFTR 5'-flanking DNA in a yeast artificial chromosome; Mogayzel PJ Jr et al.; The DNA elements that account for the highly regulated expression of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are poorly understood . The goal of this study was to assess the feasibility of using a yeast artificial chromosome (YAC)-based reporter gene construct to define these elements further . An approximately 350-kb YAC (y5'luc) was constructed by replacing CFTR with a luciferase reporter gene (luc) . A second YAC (y5'lucI) was similarly constructed but included a putative positive regulatory element from CFTR intron 1 . Stable Chinese hamster ovary (CHO-K1) cell clones were derived using each YAC to assess the role that luc copy number and the presence of intron 1 played in luc expression . The CHO-K1 clonal cell lines demonstrated a wide range of luciferase activity . On average, this activity was significantly higher in clones derived from y5'lucI . After correcting for luc copy number, the presence of intron 1 was still associated with an increase in luciferase activity (P < 0.05), despite the fact that luciferase activity did not correlate with luc copy number in y5'luc-derived clones (r = -0.12) . In contrast, the luciferase activity correlated well with luc copy number in the clones derived from y5'luc (r = 0 . 75) . These data are consistent with a positive role for intron 1 in regulating CFTR expression, but suggest that copy number is not the only factor that determines expression levels, particularly when this element is present . This YAC-based reporter system will provide a unique strategy for further assessment of the cis-acting elements that control CFTR expression .

Biochem J, 2000 Mar 1, 346 Pt 2, 329 - 36
Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter; Gitan RS et al.; The yeast ZRT1 zinc transporter is regulated by zinc at both transcriptional and post-translational levels . At the post-translational level, zinc inactivates ZRT1 by inducing the removal of the protein from the plasma membrane by endocytosis . The zinc transporter is subsequently degraded in the vacuole . This regulatory system allows for the rapid shut off of zinc uptake activity in cells exposed to high zinc concentrations, thereby preventing overaccumulation of this potentially toxic metal . In this report, we examine the role of ubiquitin conjugation in this process . First, we show that ZRT1 is ubiquitinated shortly after zinc treatment and before endocytosis . Secondly, mutations in various components of the ubiquitin conjugation pathway, specifically the RSP5 ubiquitin-protein ligase and the UBC4 and UBC5 ubiquitin conjugating enzymes, inhibit both ubiquitination and endocytosis . Finally, mutation of a specific lysine residue in ZRT1 blocks both ubiquitination and endocytosis . This critical lysine, Lys-195, is located in a cytoplasmic loop region of the protein and may be the residue to which ubiquitin is attached . These results demonstrate that ubiquitin conjugation is a critical step in the signal transduction pathway that controls the rate of ZRT1 endocytosis in response to zinc.

Biochimie, 2000 Feb, 82(2), 139 - 45
Dimerization of the Epstein-Barr virus ZEBRA protein in the yeast two-hybrid system . Comparison Of a ZEBRA variant with the B95-8 form; Martel-Renoir D et al.; Epstein-Barr virus (EBV) is a herpes virus associated with several human tumors . The EBV protein, ZEBRA, is a transactivator of the basic leucine zipper family (bZip) . It binds to specific sequences on DNA and is able to interact with cellular proteins such as p53 . The interaction of the ZEBRA protein with its cognate DNA sequences is stable as long as the dimerization domain is functional . Recent work from this laboratory identified a ZEBRA variant (Z206) with a single amino acid change at residue 206 . An alanine is substituted for a serine, and this replacement is present in 72% of nasopharyngeal carcinoma from Europe and North Africa . As amino acid 206 lies within the dimerization domain it could be instrumental in interactions with other proteins . The yeast two-hybrid system was used to study ZEBRA-protein interactions . As ZEBRA by itself is a transactivator in yeast, it cannot be used directly in this assay . This paper describes modifications in ZEBRA amino acid sequences, rendering it usable in the yeast two-hybrid assay . We compared the dimerization capacity of the Z206 variant to that of ZEBRA from B95-8 (Z95) and observed that reporter gene activity with Z206 was consistently lower than that of Z95 (P < 0.05) . Furthermore, no interaction was found to occur between either form of ZEBRA (Z206 or Z95) and the tumor suppressor, p53 in the yeast two-hybrid system.

J Dent Res, 2000 Feb, 79(2), 732 - 9
Molecular mapping of statherin- and histatin-binding domains in human salivary mucin MG1 (MUC5B) by the yeast two-hybrid system; Iontcheva I et al.; MGI is a high-molecular-weight mucin secreted by mucous acinar cells in human submandibular and sublingual glands . We have recently shown that the tracheobronchial mucin MUC5B is a major component of MG1 . MUC5B is organized into cysteine-rich N- and C-terminal regions that flank a central tandem-repeat region containing cysteine-rich subdomains and imperfect 29-residue tandem repeats . In earlier work, we have shown that this mucin selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins in salivary secretions, and the aim of this study was to identify specific binding domains within MUC5B using the yeast two-hybrid system . Interactions of cysteine-rich domains in the tandem-repeat region (Cys1-Cys4) and C-terminal region (Cys8a, Cys8b, Cys8c) of MUC5B with statherin and histatins were investigated . These studies indicated that histatin 1 selectively bound to Cysl and Cys2, whereas statherin and histatin 1, 3, and 5 selectively bound to Cys8a . Analysis of the primary sequences of the identified binding domains suggests that these domains most probably can fold into globular-like structures in the native mucin . A ProDom blast search revealed that sequences in Cys1, Cys2, and Cys8a exhibit similarity to domains in evolutionarily diverse extracellular proteins known to participate in a wide variety of protein-protein interactions.

Biochemistry, 2000 Mar 28, 39(12), 3185 - 91
Identification of 12-lipoxygenase interaction with cellular proteins by yeast two-hybrid screening; Tang K et al.; The platelet isoform of 12-lipoxygenase (12-LOX) is expressed in a variety of human tumors . 12-LOX metabolizes arachidonic acid to 12(S)-hydroxyeicosateraenoic acid (12(S)-HETE), which induces a number of cellular responses associated with tumor progression and metastasis . Little is known about 12-LOX regulation and no direct regulators of 12-LOX activity have been identified . To identify potential regulators of 12-LOX, we isolated cDNAs encoding 12-LOX interacting proteins using the yeast two-hybrid system . We screened a yeast two-hybrid interaction library from human epidermoid carcinoma A431 cells and identified four cellular proteins that interact specifically with 12-LOX . We identified type II keratin 5, lamin A, the cytoplasmic domain of integrin beta4 subunit and a phosphoprotein C8FW as 12-LOX interacting proteins . Here, we demonstrated that keratin 5, a 58 kD protein required for formation of 8 nm intermediate filaments, binds to 12-LOX in human tumor cells and may contribute to the regulated trafficking of 12-LOX . We also showed that lamin A binds 12-LOX in human tumor cells . These proteins provide the first candidate regulators of 12-LOX.

Ukr Biokhim Zh, 1999 Sep-Oct, 71(5), 104 - 9
{Characteristics of biologically active mannan substances, isolated from fodder yeast}; Kovalenko AG et al.; The component composition and antiviral properties of the mannan-containing preparations were studied . These preparations were extracted from some laboratory and commercial specimens of the fodder yeast cultivated on different substrates . It was shown, that the main component of pure preparation was the mannan which had molecular mass near 13 kDa . The monosaccaride composition of mannan component was varied depending on the source, degree of purification, methods of purification of the preparations . However, the crude and pure mannan preparations activity relatively to VTV was approximately equal.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3219 - 23
Signal transduction by a nondissociable heterotrimeric yeast G protein; Klein S et al.; Many signal transduction pathways involve heterotrimeric G proteins . The accepted model for activation of heterotrimeric G proteins states that the protein dissociates to the free G(alpha) (GTP)-bound subunit and free G(betagamma) dimer . On GTP hydrolysis, G(alpha) (GDP) then reassociates with G(betagamma) {Gilman, A . G . (1987) Annu . Rev . Biochem . 56, 615-649} . We reexamined this hypothesis, by using the mating G protein of the yeast Saccharomyces cerevisiae encoded by the genes GPA1, STE4, and STE18 . In the absence of mating pheromone, the G(alpha) (Gpa1) subunit represses the mating pathway . On activation by binding of pheromone to a serpentine receptor, the G(betagamma) (Ste4, Ste18) dimer transmits the signal to a mitogen-activated protein kinase cascade, leading to gene activation, arrest in the G(1) stage of the cell cycle, production of shmoos (mating projections), and cell fusion . We found that a Ste4-Gpa1 fusion protein transmitted the pheromone signal and activated the mating pathway as effectively as when Ste4 (G(beta)) and Gpa1 (G(alpha)) were coexpressed as separate proteins . Hence, dissociation of this G protein is not required for its activation . Rather, a conformational change in the heterotrimeric complex is likely to be involved in signal transduction.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3696 - 701
Activation of the human estrogen receptor by the antiestrogens ICI 182,780 and tamoxifen in yeast genetic systems: implications for their mechanism of action; Dudley MW et al.; The antiestrogens tamoxifen and ICI 182,780 have been portrayed as competitive antagonists of the estrogen binding site of the alpha-form of the human estrogen receptor (ER) . However, in functional studies, neither compound has consistently been able to block estradiol-induced transcription . In this report, three yeast genetic systems were used to investigate the effects of tamoxifen and ICI 182,780 on ER dimerization, transcriptional activation, and the interaction of the receptor with a coactivator, RIP140 . Tamoxifen and ICI 182,780 were able to induce ER dimerization and ER-dependent transcription, albeit at up to 15,000-fold higher concentrations than that of estradiol . In the presence of RIP140, the transcription response maximum was increased up to 30-fold for estradiol and both antiestrogens . Whole yeast cell {(3)H}estradiol binding studies demonstrated that tamoxifen could displace the estradiol from the ER, whereas ICI 182,780 treatment resulted in a 4-fold increase in {(3)H}estradiol binding to the receptor . No antagonism of estradiol was observed with tamoxifen or ICI 182,780 in any of the yeast models employed . We have concluded that the antiestrogen activity of compounds like tamoxifen and ICI 182,780 is not caused by their ability to competitively antagonize estradiol binding to the hormone binding site, but possibly by their ability to induce ER-dependent transcription, which in mammalian systems would result in receptor down-regulation . Compounds such as tamoxifen act through the hormone binding site, whereas ICI 182,780 may cause receptor activation through an allosteric binding site.

J Cell Biol, 2000 Mar 20, 148(6), 1231 - 8
The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein; Price A et al.; The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown . We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles . This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming . The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles . Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct association with a Rab/Ypt protein to activate docking.

J Cell Sci, 2000 Apr, 113 ( Pt 8), 1447 - 58
A fission yeast general translation factor reveals links between protein synthesis and cell cycle controls; Grallert B et al.; In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects in B-cyclin function or expression . These mutations define a single gene which we call ded1 . We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species . Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex . When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 (and to a lesser extent Cdc13), is impaired relative to other proteins . We show that B-cyclin translation is specifically inhibited upon nitrogen starvation of wild-type cells, when B-cyclin/Cdc2 inactivation is a prerequisite for G(1) arrest and subsequent mating . Our data suggest that translational inhibition of B-cyclin expression represents a third mechanism, in addition to cyclin degradation and Rum1 inhibition, that contributes to Cdc2 inactivation as cells exit from the mitotic cell cycle and prepare for meiosis.

Res Microbiol, 2000 Jan-Feb, 151(1), 43 - 52
At acidic pH, the diminished hypoxic expression of the SRP1/TIR1 yeast gene depends on the GPA2-cAMP and HOG pathways; Bourdineaud JP; The hypoxic SRP1/TIR1 gene encodes a stress-response cell wall mannoprotein, which is shown to be necessary for yeast growth at acidic pH in the presence of sodium dodecyl sulfate . However, the hypoxic expression of SRP1 is shown to be downregulated at acidic pH . The stress-responsive HOG pathway appeared necessary to maintain hypoxic SRP1 expression, but only at acidic pH . However, unlike known HOG pathway-dependent genes, SRP1 was under positive cAMP control and was positively modulated by protein kinase A at neutral and acidic pH . In addition, the HOG-independent hypoxic HEM13 gene was also positively regulated by cAMP levels . Therefore, the positive cAMP control of the hypoxic SRP1 and HEM13 genes was uncoupled from the HOG pathway . Surprisingly, this positive cAMP control was found to be mediated by GPA2 but not by RAS2, so the Gpa2p requirement appears critical at acidic pH . Although RAS2 is not involved in the regulation of SRP1 expression, the guanine nucleotide exchange factor Cdc25, which is known to control the GTP/GDP ratio on the Ras proteins, was nevertheless required for hypoxic SRP1 expression . Furthermore, the Ras proteins did not compensate for Gpa2p requirement in a delta gpa2 mutated strain . These results suggest that the Cdc25 factor might also control Gpa2p.

J Biol Chem, 2000 Mar 24, 275(12), 9035 - 42
Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor; Then Bergh F et al.; Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively . In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR . Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity . However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter . Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site . In contrast, GR had little effect on the stability of the MMTV B nucleosome . A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome . Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure . Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.

J Biol Chem, 2000 Mar 24, 275(12), 8382 - 8
Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway; Ciufo LF et al.; The p24 proteins are transmembrane proteins of the endomembrane system that play a poorly defined role in vesicle traffic between the endoplasmic reticulum and the Golgi apparatus . Various lines of evidence indicate that p24 proteins fall into four subfamilies (alpha, beta, gamma, and delta) and that tetramers are assembled containing one representative from each subfamily; however, the nature of the protein-protein interactions within these hetero-oligomers is unknown . We have identified a lumenal segment of yeast p24beta (Emp24p) that is necessary for its assembly into p24 complexes . Replacement of 52 C-terminal residues of Emp24p with the corresponding sequence from Erv25p (p24delta) generates a chimeric protein able to replace Emp24p in p24 complexes that retain partial function in vivo, ruling out a role for the transmembrane and cytosolic domains in specifying p24 interactions . Substitution of a further 50 residues, encompassing a heptad repeat region, abolishes the ability of the chimera to replace Emp24p but instead creates a protein that resembles its Erv25p parent in its requirement for stabilization by Emp24p . These data point to a role for coiled-coil interactions in directing subfamily-specific assembly of p24 oligomers that project into the lumen of transport vesicles, where they may act to exclude secretory cargo from coat protein complex type I-coated retrograde transport vesicles.

Toxicol Lett, 2000 Mar 15, 112-113, 239 - 44
Mutator phenotype due to loss of heterozygosity in diploid yeast strains with mutations in MSH2 and MLH1; Drotschmann K et al.; Mutations in mismatch repair (MMR) genes predispose humans to cancer . Particularly prevalent are frameshift and point mutations in MSH2 and MLH1, two genes whose products are required for the early steps in MMR . In normal tissues of persons predisposed to hereditary non-polyposis colon cancer (HNPCC), these mutations are usually present in only one allele . In tumor cells of these patients, the second, wild type allele is typically found to be deleted or inactivated by point mutation . This suggests that loss of heterozygosity (LOH) results in a strong mutator phenotype that could eventually lead to the onset of disease . Here we demonstrate that diploid yeast strains that are heterozygous for MSH2 and MLH1 alleles have an elevated mutation rate . We further show that this effect results not from saturation of the MMR capacity of all cells in the population, but rather from loss of the wild type allele in a subpopulation of heterozygous cells . These results have implications for understanding the mechanisms of carcinogenesis in humans.

EMBO J, 2000 Mar 15, 19(6), 1357 - 65
Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast; van Hoof A et al.; The biogenesis of a number of RNA species in eukaryotic cells requires 3' processing . To determine the enzymes responsible for these trimming events, we created yeast strains lacking specific 3' to 5' exonucleases . In this work, we describe the analysis of three members of the RNase D family of exonucleases (Rex1p, Rex2p and Rex3p) . This work led to three important conclusions . First, each of these exonucleases is required for the processing of distinct RNAs . Specifically, Rex1p, Rex2p and Rex3p are required for 5S rRNA, U4 snRNA and MRP RNA trimming, respectively . Secondly, some 3' exonucleases are redundant with other exonucleases . Specifically, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and RNase P RNA, and Rex1p and the exonuclease Rrp6p have an unknown redundant essential function . Thirdly, the demonstration that the Rex proteins can affect reactions that have been attributed previously to the exosome complex indicates that an apparently simple processing step can be surprisingly complex with multiple exonucleases working sequentially in the same pathway.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3254 - 9
Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast; Bagnat M et al.; Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells . Sphingolipids also have been shown to play a role in protein sorting in yeast . Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts . We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells . Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p . Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase . That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells . Using the sec18-1 mutant we found that DIGs are present already in the ER . Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.

Gene, 2000 Mar 7, 245(1), 109 - 17
Rad25p, a DNA helicase subunit of yeast transcription factor TFIIH, is required for promoter escape in vivo; Ostapenko D et al.; The general transcription factor TFIIH is required for initial DNA unwinding and promoter escape by RNA polymerase II in vitro . We examined whether Rad25p, a DNA helicase subunit of TFIIH, mediates promoter opening and promoter escape in the yeast Saccharomyces cerevisiae . DNA unwinding was probed with an in vivo permanganate reactivity assay, in a temperature-sensitive mutant of RAD25 . The consequences of Rad25p inactivation were promoter-specific . Whereas in the TDH2 promoter permanganate reactivity was entirely abolished, the reactivity at the GAL1 and GAL10 promoter regions was only moderately affected . In the GAL genes permanganate reactivity uniformly decreased downstream of the transcription start site, indicating that progression of RNA polymerase II to this region was impaired . Our results suggest that in yeast cells, promoter opening is not sufficient for productive initiation and that Rad25p-mediated promoter escape may be a limiting step in the transcription of some promoters.

Neurosci Lett, 2000 Mar 17, 282(1-2), 65 - 8
Proteolytic processing and degradation of human presenilin-1 expressed in yeast; Song S et al.; Numerous mutations causing early-onset familial Alzheimer's disease have been identified in the presenilin-1 gene . Presenilin-1 protein is produced as a 47 kDa holoprotein and proteolytically processed to an N-terminal 28 kDa and a C-terminal 19 kDa fragments by unidentified presenilinase in mammalian cells . We have demonstrated that this proteolytic processing also occurs in yeast . We also show that degradation of C-terminal fragment of presenilin-1 is dependent of proteasomal function . This yeast system will be a good tool to identify presenilinase and to study the role of presenilin-1 in amyloid precursor protein processing.

J Biol Chem, 2000 Mar 17, 275(11), 8233 - 9
Specificity of DNA lesion bypass by the yeast DNA polymerase eta; Yuan F et al.; DNA polymerase eta (Pol(eta), xeroderma pigmentosum variant, or Rad30) plays an important role in an error-free response to unrepaired UV damage during replication . It faithfully synthesizes DNA opposite a thymine-thymine cis-syn-cyclobutane dimer . We have purified the yeast Pol(eta) and studied its lesion bypass activity in vitro with various types of DNA damage . The yeast Pol(eta) lacked a nuclease or a proofreading activity . It efficiently bypassed 8-oxoguanine, incorporating C, A, and G opposite the lesion with a relative efficiency of approximately 100:56:14, respectively . The yeast Pol(eta) efficiently incorporated a C opposite an acetylaminofluorene-modified G, and efficiently inserted a G or less frequently an A opposite an apurinic/apyrimidinic (AP) site but was unable to extend the DNA synthesis further in both cases . However, some continued DNA synthesis was observed in the presence of the yeast Pol(zeta) following the Pol(eta) action opposite an AP site, achieving true lesion bypass . In contrast, the yeast Pol(alpha) was able to bypass efficiently a template AP site, predominantly incorporating an A residue opposite the lesion . These results suggest that other than UV damage, Pol(eta) may also play a role in bypassing additional DNA lesions, some of which can be error-prone.

J Biol Chem, 2000 Mar 17, 275(11), 8143 - 53
Ubiquitination of the PEST-like endocytosis signal of the yeast a-factor receptor; Roth AF et al.; A 58-residue-long, PEST-like sequence within the yeast a-factor receptor (Ste3p) specifies the ubiquitination, endocytosis, and consequent vacuolar degradation of the receptor protein (Roth, A . F., Sullivan, D . M., and Davis, N . G . (1998) J . Cell Biol . 142, 949-961) . The present work investigates three lysyl residues that map within this sequence as the potential ubiquitin acceptor sites . Lys --> Arg substitution mutants were tested for effects on both ubiquitination and endocytosis . Results indicate that the three lysines function redundantly; a severe blockade to both ubiquitination and endocytosis is seen only for receptors having all three lysines replaced . Of the three, Lys(432) plays the predominant role; ubiquitination and turnover are significantly impaired for receptors having just the K432R mutation . CNBr fragmentation of the receptor protein, used for the physical mapping of the ubiquitin attachment sites, showed PEST-like sequence lysines to be modified both with single ubiquitin moieties as well with short multi-ubiquitin chains, two or three ubiquitins long . Thus, in addition to being the signal for ubiquitination, the Ste3p PEST-like sequence also provides the site for ubiquitin attachment . To test if this endocytosis signal functions solely for ubiquitination, we have asked if the requirement for the PEST-like sequence in endocytosis might be bypassed through pre-attachment of ubiquitin to the receptor protein . Indeed, Ste3-ubiquitin translational fusions that have a ubiquitin moiety fused to the receptor in place of the PEST-like signal do undergo rapid endocytosis and vacuolar turnover . We conclude that ubiquitin alone, with no required contribution from receptor sequences, provides the sufficient signal for initiating uptake . In addition, our results confirm conclusions originally drawn from studies with the alpha-factor receptor (Terrell, J., Shih, S., Dunn, R., and Hicke, L . (1998) Mol . Cell 1, 193-202), namely that mono-ubiquitin, and not multi-ubiquitin chains provide the primary recognition determinant for uptake . Although mono-ubiquitination suffices, our results indicate that multi-ubiquitination serves to augment the rate of uptake.

J Biol Chem, 2000 Mar 17, 275(11), 7980 - 7
A mutation in yeast topoisomerase II that confers hypersensitivity to multiple classes of topoisomerase II poisons; Dong J et al.; A mutation was constructed in the CAP homology domain of yeast topoisomerase II that resulted in hypersensitivity to the intercalating agent N-{4-(9-acridinylamino)-3-methoxy-phenyl}methanesulfonamide and the fluoroquinolone 6, 8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxyli c acid, but not to etoposide . This mutation, which changes threonine at position 744 to proline, also confers hypersensitivity to anti-bacterial fluoroquinolones . The purified T744P mutant protein had wild type enzymatic activity in the absence of drugs, and no alteration in drug-independent DNA cleavage . Enhanced DNA cleavage in the presence of N-{4-(9-acridinylamino)-3-methoxy-phenyl}methanesulfonamide and fluoroquinolones was observed, in agreement with the results observed in vivo . DNA cleavage was also seen in the presence of norfloxacin and oxolinic acid, two quinolones that are inactive against eukaryotic topoisomerase II . The hypersensitivity was not associated with heat-stable covalent complexes, as was seen in another drug-hypersensitive mutant . Molecular modeling suggests that the mutation in the CAP homology domain may displace amino acids that play important roles in catalysis by topoisomerase II and may explain the drug-hypersensitive phenotype.

J Biol Chem, 2000 Mar 17, 275(11), 7626 - 32
CCC1 suppresses mitochondrial damage in the yeast model of Friedreich's ataxia by limiting mitochondrial iron accumulation; Chen OS et al.; Deletion of YFH1 in Saccharomyces cerevisiae leads to a loss of respiratory competence due to excessive mitochondrial iron accumulation . A suppressor screen identified a gene, CCC1, that maintained respiratory function in a Deltayfh1 yeast strain regardless of extracellular iron concentration . CCC1 expression prevented excessive mitochondrial iron accumulation by limiting mitochondrial iron uptake rather than by increasing mitochondrial iron egress . Expression of CCC1 did not result in sequestration of iron in membranous compartments or cellular iron export . CCC1 expression in wild type cells resulted in increased expression of the high affinity iron transport system composed of FET3 and FTR1, suggesting that intracellular iron is not sensed by the iron-dependent transcription factor Aft1p . Introduction of AFT1(up), a constitutive allele of the iron transcription factor, AFT1, that also leads to increased high affinity iron transport did not prevent Deltayfh1 cells from becoming respiratory-incompetent . Although the mechanism by which CCC1 expression affects cytosolic iron is not known, the data suggest that excessive mitochondrial iron accumulation only occurs when cytosolic free iron levels are high.

Curr Biol, 2000 Mar 9, 10(5), 231 - 40
The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle; Drury LS et al.; BACKGROUND: Cdc28p, the major cyclin-dependent kinase in budding yeast, prevents re-replication within each cell cycle by preventing the reassembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) once origins have fired . Cdc6p is a rapidly degraded protein that must be synthesised in each cell cycle and is present only during the G1 phase . RESULTS: We found that, at different times in the cell cycle, there are distinct modes of Cdc6p proteolysis . Before Start, Cdc6p proteolysis did not require either the anaphase-promoting complex (APC/C) or the SCF complex, which mediate the major cell cycle regulated ubiquitination pathways, nor did it require Cdc28p activity or any of the potential Cdc28p phosphorylation sites in Cdc6p . In fact, the activation of B cyclin (Clb)-Cdc28p kinase inactivated this pathway of Cdc6p degradation later in the cell cycle . Activation of the G1 cyclins (Clns) caused Cdc6p degradation to become extremely rapid . This degradation required the SCF(CDC4) and Cdc28p consensus sites in Cdc6p, but did not require Clb5 and Clb6 . Later in the cell cycle, SCF(CDC4)-dependent Cdc6p proteolysis remained active but became less rapid . CONCLUSIONS: Levels of Cdc6p are regulated in several ways by the Cdc28p cyclin-dependent kinase . The Cln-dependent elimination of Cdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RCs is lost before, not concomitant with, origin firing.

Mol Cell Biol, 2000 Apr, 20(7), 2455 - 65
Genetic analysis of the YDR1-BUR6 repressor complex reveals an intricate balance among transcriptional regulatory proteins in yeast; Kim S et al.; A transcriptional repressor complex encoded by two essential genes, YDR1 and BUR6, was isolated from Saccharomyces cerevisiae and shown to be the functional counterpart of the human repressor complex Dr1-DRAP1 . To elucidate the mechanism of repression by this complex, altered forms of Ydr1 and Bur6 were studied in vitro and in vivo . Deletion of the C-terminal 41 amino acids of Ydr1 resulted in loss of repressor activity and a growth defect, suggesting that the C-terminal domain of Ydr1 functions as a potent transcriptional repressor . A screen for extragenic suppressors of a cold-sensitive ydr1 (ydr1(cs)) mutant led to the identification of recessive mutations in the SIN4 gene, which encodes a component of the SRB-MED complex . The sin4 alleles suppressed not only ydr1(cs) mutations but also bur6(cs) mutations . In contrast, deletion of the gal11 gene, whose product is also a member of the SRB-MED complex, failed to suppress ydr1(cs) and bur6(cs) mutations, indicating that suppression is not due to general defects in the SRB-MED complex . Moreover, one of the sin4 alleles, but not the sin4 deletion, was found to specifically suppress the inviability of a ydr1 deletion, demonstrating that the essential function of Ydr1 becomes dispensable in a sin4 mutant background . Biochemical analysis of the SRB-MED complex from the sin4 suppressor strain revealed a structurally distinct form of the SRB-MED complex that lacks a subset of mediator subunits . These results define a delicate balance between positive and negative regulators of transcription operating through the Ydr1-Bur6 repressor complex.

Mol Cell Biol, 2000 Apr, 20(7), 2385 - 99
Impaired core promoter recognition caused by novel yeast TAF145 mutations can be restored by creating a canonical TATA element within the promoter region of the TUB2 gene; Tsukihashi Y et al.; The general transcription factor TFIID, which is composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs), has been shown to play a crucial role in recognition of the core promoters of eukaryotic genes . We isolated Saccharomyces cerevisiae yeast TAF145 (yTAF145) temperature-sensitive mutants in which transcription of a specific subset of genes was impaired at restrictive temperatures . The set of genes affected in these mutants overlapped with but was not identical to the set of genes affected by a previously reported yTAF145 mutant (W.-C . Shen and M . R . Green, Cell 90:615-624, 1997) . To identify sequences which rendered transcription yTAF145 dependent, we conducted deletion analysis of the TUB2 promoter using a novel mini-CLN2 hybrid gene reporter system . The results showed that the yTAF145 mutations we isolated impaired core promoter recognition but did not affect activation by any of the transcriptional activators we tested . These observations are consistent with the reported yTAF145 dependence of the CLN2 core promoter in the mutant isolated by Shen and Green, although the CLN2 core promoter functioned normally in the mutants we report here . These results suggest that different promoters require different yTAF145 functions for efficient transcription . Interestingly, insertion of a canonical TATA element into the TATA-less TUB2 promoter rescued impaired transcription in the yTAF145 mutants we studied . It therefore appears that strong binding of TBP to the core promoter can alleviate the requirement for at least one yTAF145 function.

Mol Cell Biol, 2000 Apr, 20(7), 2367 - 77
The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein; Li SJ et al.; Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins . Like SMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein . We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2 . In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1(ts) strain, which is defective for a distantly related Smt3-specific protease, Ulp1 . The ulp2Delta mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect . The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl . Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired . Surprisingly, either introduction of a ulp1(ts) mutation or overproduction of catalytically inactive Ulp1 can substantially overcome the ulp2Delta defects . Inactivation of Ulp2 also suppresses several ulp1(ts) defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant . Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function.

Mutat Res, 2000 Feb 16, 459(1), 55 - 64
Mitotic viability and metabolic competence in UV-irradiated yeast cells; Conconi A et al.; Colony formation is the classic method for measuring survival of yeast cells . This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events . Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation . The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta) . Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells . However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation . Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate . For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA . Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period . In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity) . These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.

Mol Cell, 2000 Jan, 5(1), 163 - 72
Rnq1: an epigenetic modifier of protein function in yeast; Sondheimer N et al.; Two protein-based genetic elements (prions) have been identified in yeast . It is not clear whether other prions exist, nor is it understood how one might find them . We established criteria for searching protein databases for prion candidates and found several . The first examined, Rnq1, exists in distinct, heritable physical states, soluble and insoluble . The insoluble state is dominant and transmitted between cells through the cytoplasm . When the prion-like region of Rnq1 was substituted for the prion domain of Sup35, the protein determinant of the prion {PSI+}, the phenotypic and epigenetic behavior of {PSI+} was fully recapitulated . These findings identity Rnq1 as a prion, demonstrate that prion domains are modular and transferable, and establish a paradigm for identifying and characterizing novel prions.

Appl Microbiol Biotechnol, 2000 Feb, 53(2), 152 - 8
Synthesis of alpha-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol; Chernyavskaya OG et al.; The ability of yeast to synthesize alpha-ketoglutaric acid (KGA) from ethanol has been studied . Thiamine-auxotrophic yeasts of different genera and species may be able to produce KGA; the main condition of synthesis is growth limitation by thiamine . Using a model culture, mutant Yarrowia lipolytica N 1, the principal conditions affecting KGA oversynthesis were identified . These were: thiamine concentration in medium and in cells, nitrogen and oxygen concentration in medium, and pH level . A KGA concentration of 49 g/l and a yield from ethanol consumed of 42% were achieved . Based on the results of the analysis of the activities of the key enzymes participating in ethanol metabolism and KGA synthesis, a concept of the mechanism of KGA biosynthesis by Y . lipolytica yeast is suggested and discussed.

Biochem Biophys Res Commun, 2000 Mar 16, 269(2), 474 - 80
Identification of a family of noncanonical ubiquitin-conjugating enzymes structurally related to yeast UBC6; Lester D et al.; Ubiquitin-conjugating enzymes (UBCs) selectively target proteins for proteasomal degradation by the covalent attachment of ubiquitin moieties . Yeast UBC6 is unusual in having an active site distinct from all other UBCs and in possessing a transmembrane domain that anchors it to the cytoplasmic surface of the endoplasmic reticulum . During a differential display analysis on chick growth plate chondrocytes we isolated a cDNA encoding a noncanonical ubiquitin-conjugating enzyme (NCUBE1) structurally similar to yeast UBC6 . Chick NCUBE1 transcripts were detected in all tissues examined and decreased threefold during chondrocyte terminal differentiation . Database searches identified other related proteins; the human and mouse orthologues of NCUBE1, a second human homologue of yeast UBC6 (NCUBE2), and related proteins from S . pombe, C . elegans, and P . mariana . Together with yeast UBC6 these proteins constitute a distinct family of UBCs sharing a conserved noncanonical active site sequence and a C-terminal transmembrane domain . By analogy with yeast UBC6 they are likely to be localised to the endoplasmic reticulum where they may be involved in targeting retrotranslocated, ER-associated proteins for proteasomal degradation .

Nucleic Acids Res, 2000 Apr 1, 28(7), 1656 - 64
In vivo, high-resolution analysis of yeast and mammalian RNA-protein interactions, RNA structure, RNA splicing and ribozyme cleavage by use of terminal transferase-dependent PCR; Chen HH et al.; We have investigated the analysis of RNA by use of terminal transferase-dependent PCR (TDPCR), a procedure previously used for the analysis of DNA and chromatin {J.Komura and A.D.Riggs, Nucleic Acids Res.,26, 1807-1811 (1998)} . When preceded by reverse transcription (RT), TDPCR provides an extremely sensitive, versatile, quantitative and nucleotide-level assay for detecting RNA lesions or structures that block primer extension during the RT step . The procedure is: (i) RT using a gene-specific oligonucleotide; (ii) ribo-tailing of the single-stranded cDNA product by use of terminal deoxy-nucleotidyl transferase; (iii) ligation of a DNA linker to the tailed cDNA by use of T4 DNA ligase; and (iv) PCR using a nested, gene-specific primer and a linker-specific primer . This procedure combines the versatility of a primer extension assay with nucleotide-level resolution, the specificity of nested primers and the sensitivity of PCR . Band patterns obtained are reproducible and quantifiable . We successfully used the technique for the study of yeast RNA structure, splicing intermediates and ribozyme cleavage . Also, in vivo footprint experiments, using mammalian cells and RNase T1, revealed the binding of iron-responsive element binding protein to iron responsive elements in the mRNAs of transferrin receptor and ferritin H-chain.

Nucleic Acids Res, 2000 Apr 1, 28(7), 1594 - 603
Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system; Martin F et al.; We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants . Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198 . Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes . These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure . Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD . Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD.

Nucleic Acids Res, 2000 Apr 1, 28(7), 1576 - 84
Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast; Lang BD et al.; Scp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains 14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant sequence homology to a family of proteins collectively known as vigilins . As a first step towards defining the function of Scp160p, we have characterized the subcellular distribution and in vivo interactions of this protein . Using sucrose gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic lysates is predominantly associated with polyribosomes . Furthermore, we have found that Scp160p is released from polyribosomes by EDTA in the form of a large complex of> or =1300 kDa that is sensitive both to RNase and NaCl . Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p . The presence of Pab1p confirms these complexes to be mRNPs . The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160 -null cells: increased cell size and aberrant DNA content . These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast.

Microbiology, 2000 Feb, 146 ( Pt 2), 385 - 91
The yeast Chs4 protein stimulates the trypsin-sensitive activity of chitin synthase 3 through an apparent protein-protein interaction; Ono N et al.; Inducible overexpression of the CHS4 gene under the control of the GAL1 promoter increased Chs3p (chitin synthase 3) activity in Saccharomyces cerevisiae several fold . Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate . The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP-N-acetylglucosamine . Western blotting confirmed that Chs3p was extracted from membranes by CHAPS and cholesteryl hemisuccinate and revealed that Chs4p was also solubilized using these detergents . Yeast two-hybrid analysis with truncated Chs4p demonstrated that the region of Chs4p between amino acids 269 and 563 is indispensable not only for eliciting the non-zymogenic activity of Chs3p but also for binding of Chs4p to Chs3p . Neither the EF-hand motif nor a possible prenylation site in Chs4p was required for these activities . Thus, it was demonstrated that stimulation of non-zymogenic Chs3p activity by Chs4p requires the amino acid region from 269 to 563 of Chs4p, and it seems that Chs4p activates Chs3p through protein-protein interaction.

Biosci Biotechnol Biochem, 2000 Jan, 64(1), 89 - 95
Requirement for lysine-19 of the yeast mitochondrial ATPase inhibitor for the stability of the inactivated inhibitor-F1Fo complex at higher pH; Ichikawa N et al.; The ATPase inhibitor is a regulatory subunit of mitochondrial ATP synthase . In this study, the role of Lys19 of the yeast ATPase inhibitor was examined by site-directed mutagenesis . Two amino acids (Gln and Glu) were substituted for the Lys19 . The purified mutant inhibitor (Lys19-->Gln) had similar ATPase inhibitory activity to that of the wild-type inhibitor at pH 6.5, but was less active at pH 7.4 . ATP synthesis in mutant mitochondria was normally activated by the addition of ADP and succinate, but the inactivated ATPase complex in the mutant mitochondria was activated more readily than that in control cells by raising pH . These results show that Lys19 of the yeast ATPase inhibitor is not essential for ATPase inhibitory activity, but increases the stability of the inhibitor-F1Fo complex at higher pH.

Diagn Microbiol Infect Dis, 2000 Feb, 36(2), 137 - 8
Rapid identification of clinical yeast isolates using the colorimetric AUXACOLOR system; Romney MG et al.; The AUXACOLOR colorimetric system (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) for the identification of clinical yeast isolates, was compared in its identification of 100 yeast strains to conventional identification methods . Of the 94 correctly identified isolates, 47% (n = 44) were identified by 24 h, and 100% (n = 94) were identified by 48 h . AUXACOLOR is a simple, rapid and accurate method for the identification of yeast pathogens.

J Steroid Biochem Mol Biol, 1999 Dec 31, 71(5-6), 239 - 46
Pregnenolone metabolized to 17alpha-hydroxyprogesterone in yeast: biochemical analysis of a metabolic pathway; Degryse E et al.; The cDNA coding for the human 3beta-hydroxy-5-ene steroid dehydrogenase/5-ene-4-ene steroid isomerase (3beta-HSD) has been expressed in yeast . When expressed from identical vectors except for the coding sequence, the specific activity of the type I is lower than that of the type II enzyme . A mutant of the human 3beta-HSD type II lacking the putative membrane spanning domain 1 was generated by site directed mutagenesis: its apparent K(m) for pregnenolone (PREG) is significantly increased and its V reduced to the level of the type I enzyme . The influence of the kinetic properties of 3beta-HSD in the accumulation of 17alpha-hydroxyprogesterone was probed by co-expression of the bovine 17alpha-hydroxylase cytochrome P450 (P45017alpha) cDNA . The metabolism of PREG was followed with time using the membrane fraction . Kinetic properties of the 3beta-HSD were modulated such that its activity was in excess, limiting or balanced with respect to the activity of the P45017alpha and the accumulation of intermediates and products recorded . Conditions for the generation of the by-products resulting from the 17,20-Lyase activity of the P45017alpha were found . The potential applications of the system are discussed.

Biotechnol Bioeng, 2000 Apr 5, 68(1), 44 - 51
Efficient bioconversion of ethanol to acetaldehyde using a novel mutant strain of the methylotrophic yeast Hansenula polymorpha; Moroz OM et al.; We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system . The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H . polymorpha cells growing on methanol . At least three mutations were introduced in the strains . Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast . The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate . Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined . In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin . Under optimal shake-flask conditions using one of the H . polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative .

J Pharm Biomed Anal, 1999 Feb, 19(1-2), 115 - 25
Using entropies of reaction to predict changes in protein stability: tyrosine-67-phenylalanine variants of rat cytochrome c and yeast Iso-1 cytochromes c; Feinberg BA et al.; Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c . In addition to determining the equilibrium reduction potential (E0') for each cytochrome, the entropy of reaction, deltaS0'(Rxn)(deltaS0'(Rxn) = S0'(Red) - S0'(Ox)), for the reduction process was determined via the non-isothermal method . Having determined deltaS0'(Rxn) and E0', deltaH0' was calculated . For rat cytochrome c, it was found that deltaS0'(Rxn) = -43 J mol(-1) K(-1) for the wild type and -53 J mol(-1) K(-1) for the Tyr67Phe variant, with the deltaH0' for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in deltaS0'(Rxn) . In contrast the measured deltaS0'(Rxn) for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c . The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein . A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein . For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements . While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant.

Antonie Van Leeuwenhoek, 2000 Jan, 77(1), 43 - 7
The isolation and classification of Candida mokoenaii sp . nov . A new yeast isolate from South African soil; Mokwena TA et al.; A new xylanase producing yeast species was isolated from South African soil . A description of the new species, Candida mokoenaii is given.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1835 - 40
An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins; O'Regan S et al.; Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine . We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation . The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast . The gene was called CTL1 for its choline transporter-like properties . The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3 . 5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon . In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain . High levels of rCTL1 were also identified in the mucosal cell layer of the colon . Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2-4) . These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1589 - 94
Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins; Krobitsch S et al.; Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein . A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons . Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear . To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein . The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation . Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation . Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation . However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments . More dramatically, the deletion of Hsp104 virtually eliminated it . These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1516 - 20
Studies on the role of the hydrophobic domain of Ost4p in interactions with other subunits of yeast oligosaccharyl transferase; Kim H et al.; In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT), which catalyzes the transfer of dolichol-linked oligosaccharide chains to nascent polypeptides in the endoplasmic reticulum, consists of nine nonidentical membrane protein subunits . Genetic and biochemical evidence indicated these nine proteins exist in three subcomplexes . Three of the OT subunits (Ost4p, Ost3p, and Stt3p) have been proposed to exist in one subcomplex . To investigate the interaction of these three membrane proteins, initially we carried out a mutational analysis of Ost4p, which is an extraordinarily small membrane protein containing only 36 amino acid residues . This analysis indicated that when single amino acid residues in a region close to the luminal face of the putative transmembrane domain of Ost4p were changed into an ionizable amino acid such as Lys or Asp, growth at 37 degrees C and OT activity measured in vitro were impaired . In addition, using immunoprecipitation techniques and Western blot analysis, we found that with these mutations the interaction between Ost4p, Ost3p, and Stt3p was disrupted . Introduction of Lys or Asp residues at other positions in the putative transmembrane domain or at the N or C terminus of Ost4p had no effect on disrupting subunit interactions or impairing the activity of OT . These findings suggest that a localized region of the putative transmembrane domain of Ost4p mediates in stabilization of the interaction with the two other OT subunits (Ost3p and Stt3p) in a subcomplex in the endoplasmic reticulum membrane.

J Biol Chem, 2000 Mar 10, 275(10), 6963 - 8
Partial uncoupling of the mitochondrial membrane by a heterozygous null mutation in the gene encoding the gamma- or delta-subunit of the yeast mitochondrial ATPase; Xiao Y et al.; Prior genetic studies indicated that the yeast mitochondrial ATP synthase can be assembled into enzyme complexes devoid of the gamma-, delta-, or epsilon-subunits (Lai-Zhang, J., Xiao, Y., and Mueller, D . M . (1999) EMBO J . 18, 58-64) . These subunit-deficient complexes were postulated to uncouple the mitochondrial membrane thereby causing negative cellular phenotypes . This study provides biochemical and additional genetic data that support this hypothesis . The genetic data indicate that in a diploid cell, a heterozygous deletion mutation in the gene encoding the gamma- or delta-subunit of the ATPase is semidominant negative due to a decrease in the gene number from 2 to 1 . However, the heterozygous atp2Delta mutation is epistatic to the heterozygous mutation in the gene encoding gamma or delta, suggesting that the semidominant negative effect is because of a gain of activity in the cells . Biochemical studies using mitochondria isolated from the yeast strains that are heterozygous for a mutation in gamma or delta indicate that the mitochondria are partially uncoupled . These results support the hypothesis that the negative phenotypes are caused by the formation of a gamma- or delta-less ATP synthase complex that is uncoupled.

J Biol Chem, 2000 Mar 10, 275(10), 6707 - 11
Cell division regulation by BIR1, a member of the inhibitor of apoptosis family in yeast; Li F et al.; The inhibitor of apoptosis (IAP) gene family comprises molecules that block the activity of pro-apoptotic caspase proteases . Paradoxically, yeasts contain IAP proteins but no caspases and no apoptotic program . To determine the function of these proteins in vivo, we disrupted the BIR1 gene, encoding the only known IAP in yeast Saccharomyces cerevisiae . Sporulation of heterozygous diploids yielded no viable mutant haploids, indicating that BIR1 is an essential gene . By flow cytometry, some heterozygous mutants were polyploid accumulating >4 N DNA content . These cells exhibited a 20-40% reduction in growth rate, which was rescued by plasmid-borne over-expression of BIR1 but not by its human counterpart, survivin . Deletion analysis revealed that the N-terminal domain of Bir1, containing the conserved baculovirus IAP repeat, was able to partially complement the cell growth defect caused by BIR1 deletion . Moreover, the full-length and truncated forms of Bir1 accelerated cell division in wild-type cells . Finally, BIR1 heterozygous mutants exhibited grossly altered cell morphology with misshapen or abnormally long buds connected to an unusually large mother cell . These findings identify a novel function of IAP proteins in the pleiotropic control of cell division, in addition to their role in the suppression of apoptosis.

Biochem J, 2000 Jan 15, 345 Pt 2, 321 - 34
Effect of cellular interaction on glycolytic oscillations in yeast: a theoretical investigation; Wolf J et al.; On the basis of a detailed model of yeast glycolysis, the effect of intercellular dynamics is analysed theoretically . The model includes the main steps of anaerobic glycolysis, and the production of ethanol and glycerol . Transmembrane diffusion of acetaldehyde is included, since it has been hypothesized that this substance mediates the interaction . Depending on the kinetic parameter, the single-cell model shows both stationary and oscillatory behaviour . This agrees with experimental data with respect to metabolite concentrations and phase shifts . The inclusion of intercellular coupling leads to a variety of dynamical modes, such as synchronous oscillations, and different kinds of asynchronous behavior . These oscillations can co-exist, leading to bi- and tri-rhythmicity . The corresponding parameter regions have been identified by a bifurcation analysis . The oscillatory dynamics of synchronized cell populations are investigated by calculating the phase responses to acetaldehyde pulses . Simulations are performed with respect to the synchronization of two subpopulations that are oscillating out of phase before mixing . The effect of the various process on synchronization is characterized quantitatively . While continuous exchange of acetaldehyde might synchronize the oscillations for appropriate sets of parameter values, the calculated synchronization time is longer than that observed experimentally . It is concluded either that addition to the transmembrane exchange of acetaldehyde, other processes may contribute to intercellular coupling, or that intracellular regulator feedback plays a role in the acceleration of the synchronization . for appropriate sets of parameter values, the calculated synchronization time is longer than that observed experimentally . It is concluded either that addition to the transmembrane exchange of acetaldehyde, other processes may contribute to intercellular coupling, or that intracellular regulator feedback plays a role in the acceleration of the synchronization.

Mol Reprod Dev, 2000 Apr, 55(4), 372 - 8
Murine Myak, a member of a family of yeast YAK1-related genes, is highly expressed in hormonally modulated epithelia in the reproductive system and in the embryonic central nervous system; Shang E et al.; We have cloned a mouse homologue (designated Myak) of the yeast protein kinase YAK1 . The 1210 aa open reading frame contains a putative protein kinase domain, nuclear localization sequences and PEST sequences . Myak appears to be a member of a growing family of YAK1-related genes that include Drosophila and human Minibrain as well as a recently identified rat gene ANPK that encode a steroid hormone receptor interacting protein . RNA blot analysis revealed that Myak is expressed at low levels ubiquitously but at high levels in reproductive tissues, including testis, epididymis, ovary, uterus, and mammary gland, as well as in brain and kidney . In situ hybridization analysis on selected tissues revealed that Myak is particularly abundant in the hormonally modulated epithelia of the epididymis, mammary gland, and uterus, in round spermatids in the testis, and in the corpora lutea in the ovary . Myak is also highly expressed in the aqueduct of the adult brain and in the brain and spinal cord of day 12.5 embryos .

Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2491 - 6
The interaction of nitric oxide (NO) with the yeast transcription factor Ace1: A model system for NO-protein thiol interactions with implications to metal metabolism; Shinyashiki M et al.; Nitric oxide (NO) was found to inhibit the copper-dependent induction of the yeast CUP1 gene . This effect is attributable to an inhibition of the copper-responsive CUP1 transcriptional activator Ace1 . A mechanism is proposed whereby the metal binding thiols of Ace1 are chemically modified via NO- and O(2)-dependent chemistry, thereby diminishing the ability of Ace1 to bind and respond to copper . Moreover, it is proposed that demetallated Ace1 is proteolytically degraded in the cell, resulting in a prolonged inhibition of copper-dependent CUP1 induction . These findings indicate that NO may serve as a disrupter of yeast copper metabolism . More importantly, considering the similarity of Ace1 to other mammalian metal-binding proteins, this work lends support to the hypothesis that NO may regulate/disrupt metal homeostasis under both normal physiological and pathophysiological circumstances.

Biochemistry, 2000 Mar 7, 39(9), 2406 - 12
Effect of pressure on deuterium isotope effects of yeast alcohol dehydrogenase: evidence for mechanical models of catalysis; Northrop DB et al.; Moderate pressure accelerates hydride transfer catalyzed by yeast alcohol dehydrogenase, indicative of a large negative volume of activation {Cho and Northrop (1999) Biochemistry 38, 7470-7475} . A comparison of the effects of pressure on the oxidation of normal versus dideuteriobenzyl alcohol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 10.4 mL/mol . This finding appears consistent with hydrogen tunneling previously proposed for this dehydrogenase {Cha, Y., Murray, C . J., and Klinman, J . P . (1989) Science 243, 1325-1330} . However, a global fit of the primary data shows that the entire isotope effect arises from a transition-state phenomenon, unlike normal isotope effects, which arise from different vibrational frequencies in reactant states, and tunneling isotope effects, which arise from a mixture of both states . Assuming the phenomenon is tunneling, the isotopic data are consistent with a Bell tunneling correction factor of Q(H) = 12 and an imaginary frequency of nu(H) = 1220 cm(-1), the first so calculated from experimental enzymatic data . This excessively large correction factor and the large difference in the isotopic activation volumes, plus the low isotope effects at extrapolated pressures, challenge traditional applications of physical organic chemistry and transition-state theory to enzymatic catalysis . They suggest instead that something other than transition-state stabilization or tunneling is responsible for the rate acceleration, something unique to the enzymatic transition state that does not occur in nonenzymatic reactions . Arguments for the vibrational model of coupled atomic motions and the fluctuating enzyme model of protein domain motion are put forward as possible interpretations.

Biochemistry, 2000 Mar 7, 39(9), 2316 - 24
Spectroscopic analysis of the trinuclear cluster in the Fet3 protein from yeast, a multinuclear copper oxidase; Blackburn NJ et al.; The Fet3 protein (Fet3p) is a multinuclear copper oxidase essential for high-affinity iron uptake in yeast . Fet3p contains one type 1, one type 2, and a strongly antiferromagnetically coupled binuclear Cu(II)-Cu(II) type 3 copper . The type 2 and type 3 sites constitute a structurally distinct trinuclear cluster at which dioxygen is reduced to water . In Fet3p, as in ceruloplasmin, Fe(II) is oxidized to Fe(III) at the type 1 copper; this is the ferroxidase reaction that is fundamental to the physiologic function of these two enzymes . Using site-directed mutagenesis, we have generated type 1-depleted (T1D), type 2-depleted (T2D), and T1D/T2D mutants . None were active in the essential ferroxidase reaction catalyzed by Fet3p . However, the spectroscopic signatures of the remaining Cu(II) sites in any one of the three mutants were indistinguishable from those exhibited by the wild type . Although the native protein and the T1D mutant were isolated in the completely oxidized Cu(II) form, the T2D and T1D/T2D mutants were found to be completely reduced . This result is consistent with the essential role of the type 2 copper in dioxygen turnover, and with the suggestions that cuprous ion is the valence state of intracellular copper . Although stable to dioxygen, the Cu(I) sites in both proteins were readily oxidized by hydrogen peroxide . The double mutant was extensively analyzed by X-ray absorption spectroscopy . Edge and near-edge features clearly distinguished the oxidized from the reduced form of the binuclear cluster . EXAFS was strongly consistent with the expected coordination of each type 3 copper by three histidine imidazoles . Also, copper scattering was observed in the oxidized cluster along with scattering from a ligand corresponding to a bridging oxygen . The data derived from the reduced cluster indicated that the bridge was absent in this redox state . In the reduced form of the double mutant, an N/O ligand was apparent that was not seen in the reduced form of the T1D protein . This ligand in T1D/T2D could be either the remaining type 2 copper imidazole ligand (from His416) or a water molecule that could be stabilized at the type 3 cluster by H-bonding to this side chain . If present in the native protein, this H(2)O could provide acid catalysis of dioxygen reduction at the reduced trinuclear center.

Biochim Biophys Acta, 2000 Feb 24, 1457(1-2), 36 - 44
Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc(1) complex of yeast mitochondria; Obungu VH et al.; Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex . To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae . In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90% . No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein . EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90) . Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C . suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex . By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.

Biophys J, 2000 Mar, 78(3), 1145 - 53
Transduction of intracellular and intercellular dynamics in yeast glycolytic oscillations; Wolf J et al.; Under certain well-defined conditions, a population of yeast cells exhibits glycolytic oscillations that synchronize through intercellular acetaldehyde . This implies that the dynamic phenomenon of the oscillation propagates within and between cells . We here develop a method to establish by which route dynamics propagate through a biological reaction network . Application of the method to yeast demonstrates how the oscillations and the synchronization signal can be transduced . That transduction is not so much through the backbone of glycolysis, as via the Gibbs energy and redox coenzyme couples (ATP/ADP, and NADH/NAD), and via both intra- and intercellular acetaldehyde.

Biophys J, 2000 Mar, 78(3), 1087 - 93
How yeast cells synchronize their glycolytic oscillations: a perturbation analytic treatment; Bier M et al.; Of all the lifeforms that obtain their energy from glycolysis, yeast cells are among the most basic . Under certain conditions the concentrations of the glycolytic intermediates in yeast cells can oscillate . Individual yeast cells in a suspension can synchronize their oscillations to get in phase with each other . Although the glycolytic oscillations originate in the upper part of the glycolytic chain, the signaling agent in this synchronization appears to be acetaldehyde, a membrane-permeating metabolite at the bottom of the anaerobic part of the glycolytic chain . Here we address the issue of how a metabolite remote from the pacemaking origin of the oscillation may nevertheless control the synchronization . We present a quantitative model for glycolytic oscillations and their synchronization in terms of chemical kinetics . We show that, in essence, the common acetaldehyde concentration can be modeled as a small perturbation on the "pacemaker" whose effect on the period of the oscillations of cells in the same suspension is indeed such that a synchronization develops.

J Biol Chem, 2000 Mar 3, 275(9), 6227 - 33
Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis; Schmidt WK et al.; The yeast mating pheromone a-factor precursor contains an N-terminal extension and a C-terminal CAAX motif within which multiple posttranslational processing events occur . A recently discovered component in a-factor processing is Ste24p/Afc1p, a multispanning endoplasmic reticulum membrane protein that contains an HEXXH metalloprotease motif . Our in vivo genetic characterization of this protein has demonstrated roles for Ste24p in both the N-terminal and C-terminal proteolytic processing of the a-factor precursor . Here, we present evidence that the N-terminal proteolysis of the a-factor precursor P1 can be accurately reconstituted in vitro using yeast membranes . We show that this activity is dependent on Ste24p and is abolished by mutation of the Ste24p HEXXH metalloprotease motif or by mutation of the a-factor P1 substrate at a residue adjacent to the N-terminal P1 cleavage site . We also demonstrate that N-terminal proteolysis of the P1 a-factor precursor requires Zn(2+) as a co-factor and can be inhibited by the addition of the metalloprotease inhibitor 1,10-orthophenanthroline . Our results are consistent with Ste24p itself being the P1-->P2 a-factor protease or a limiting activator of this activity . Interestingly, we also show that the human Ste24 homolog expressed in yeast can efficiently promote the N-terminal processing of a-factor in vivo and in vitro, thus establishing a-factor as a surrogate substrate in the absence of known human substrates . The results reported here, together with the previously reported in vitro reconstitution of Ste24p-dependent CAAX processing, provide a system for examining the potential bifunctional roles of yeast Ste24p and its homologs.

Environ Mol Mutagen, 2000, 35(1), 31 - 8
UV-induced mutagenesis of human p53: analysis using a double-selection method in yeast; Moshinsky DJ et al.; Comparison of the mutation patterns of p53 in human tumors with those of selectable genes in model systems is a powerful approach to identify potential etiological factors for specific tumor types . Recently, we validated use of a yeast assay to permit direct determination of the mutation spectrum induced in human p53 by carcinogens that would reduce uncertainties inherent in comparing spectra induced in different target genes . Here, we describe modifications in the assay designed to facilitate screening for mutants and to permit intracellular exposure of the gene instead of in vitro treatment . This was accomplished by introducing growth-based selection for transactivation-deficient p53 mutants into yeast already possessing red/white colony color selection . This improved model system was able to detect cells harboring p53 mutations among cells with wild-type p53 at a frequency of 10(-4) or less . Additionally, UV light was used to verify that the majority of mutagenized cells with the appropriate phenotype on selective medium contained mutations in p53, not elsewhere in the genome . Sequence analysis of UV-induced mutations revealed that the nature of the mutations was similar to those obtained in previous studies of this mutagen . This system will prove useful in the determination of the ability of environmental agents to mutate the human p53 gene, and thus may contribute to hazard identification .

Mol Microbiol, 2000 Feb, 35(4), 825 - 34
Interactions between Pho85 cyclin-dependent kinase complexes and the Swi5 transcription factor in budding yeast; Measday V et al.; Pho85 is a cyclin-dependent protein kinase (Cdk) in budding yeast with roles in cell metabolism and cell cycle progression . Activation of Pho85 occurs through association with Pho85 cyclins (Pcls), of which 10 are known . When complexed with the G1 cyclins, Pcl1 and Pcl2, Pho85 is required for cell cycle progression in the absence of the Cdc28-dependent cyclins, Cln1 and Cln2 . To identify potential targets of Pcl2-Pho85, we performed a two-hybrid screen using the Pcl2 cyclin as bait and recovered the transcription factor Swi5 as a Pcl2-interacting protein . We performed both biochemical and genetic tests to discover the biological significance of the interaction between Pcl2 and Swi5 seen in the two-hybrid assay . We found that Swi5 interacts in vitro with Pho85 cyclins and is phosphorylated in vitro by the Pho80-Pho85 kinase . We discovered that a subset of genes that are controlled by Swi5 and a homologous transcription factor, Ace2, was misregulated in a pho85 deletion strain; expression of the ASH1 and CTS1 genes was reduced in an ace2 deletion strain, whereas expression of both genes was increased in an ace2Delta pho85Delta double mutant . We also found that overexpression of SWI5 caused cell lethality in a pho85 deletion strain . Our results are consistent with misregulation of Swi5 activity in vivo in the absence of Pho85 and implicate Swi5 as a potential substrate of Pho85 cyclin-dependent kinase complexes.

Genes Dev, 2000 Feb 15, 14(4), 452 - 63
Yeast heterochromatin is a dynamic structure that requires silencers continuously; Cheng TH et al.; Transcriptional silencing of the HM loci in yeast requires cis-acting elements, termed silencers, that function during S-phase passage to establish the silent state . To study the role of the regulatory elements in maintenance of repression, site-specific recombination was used to uncouple preassembled silent chromatin fragments from silencers . DNA rings excised from HMR were initially silent but ultimately reactivated, even in G(1)- or G(2)/M-arrested cells . In contrast, DNA rings bearing HML-derived sequence were stably repressed due to the presence of a protosilencing element . These data show that silencers (or protosilencers) are required continuously for maintenance of silent chromatin . Reactivation of unstably repressed rings was blocked by overexpression of silencing proteins Sir3p and Sir4p, and chromatin immunoprecipitation studies showed that overexpressed Sir3p was incorporated into silent chromatin . Importantly, the protein was incorporated even when expressed outside of S phase, during G(1) arrest . That silencing factors can associate with and stabilize preassembled silent chromatin in non-S-phase cells demonstrates that heterochromatin in yeast is dynamic.

Radiats Biol Radioecol, 1999 Nov-Dec, 39(6), 619 - 22
{Features of action of low doses of gamma-radiation on yeast cells}; Ziuzikov NA et al.; Lethal effect of low doses and adaptive response to low doses of prolonged irradiation were investigated in experiments on yeast cells . The phenomenon of hypersensitivity at low dozes was not found in yeast cells at gamma-irradiation . The adaptive response was observed after exposure to low doses of prolonged irradiation, the degree of the reaction depends on a dose rate . The adaptive reaction was kept for some time after the termination of adaptive irradiation then the sensitivity of cells increased even in comparison with unirradiated ones.

Mol Biol Cell, 2000 Feb, 11(2), 747 - 63
The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells; Yoshimori T et al.; The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole . To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p . Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein . This was due to loss of their receptors from the cell surface . The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein . The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1 . TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1-localized compartments . Degradation of epidermal growth factor was inhibited . Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase . The early endosome antigen EEA1 was also redistributed to these aberrant membranes . Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.

Mol Biol Cell, 2000 Feb, 11(2), 691 - 702
The yeast kinesin-related protein Smy1p exerts its effects on the class V myosin Myo2p via a physical interaction; Beningo KA et al.; We have discovered evidence for a physical interaction between a class V myosin, Myo2p, and a kinesin-related protein, Smy1p, in budding yeast . These proteins had previously been linked by genetic and colocalization studies, but we had been unable to determine the nature of their association . We now show by two-hybrid analysis that a 69-amino acid region of the Smy1p tail interacts with the globular portion of the Myo2p tail . Deletion of this myosin-binding region of Smy1p eliminates its ability to colocalize with Myo2p and to overcome the myo2-66 mutant defects, suggesting that the interaction is necessary for these functions . Further insights about the Smy1p-Myo2p interaction have come from studies of a new mutant allele, myo2-2, which causes a loss of Myo2p localization . We report that Smy1p localization is also lost in the myo2-2 mutant, demonstrating that Smy1p localization is dependent on Myo2p . We also found that overexpression of Smy1p partially restores myo2-2p localization in a myosin-binding region-dependent manner . Thus, overexpression of Smy1p can overcome defects in both the head and tail domains of Myo2p (caused by the myo2-66 and myo2-2 alleles, respectively) . We propose that Smy1p enhances some aspect of Myo2p function, perhaps delivery or docking of vesicles at the bud tip.

Mol Biol Cell, 2000 Feb, 11(2), 613 - 26
VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment; Gerrard SR et al.; Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole . The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport . Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes . Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar . We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole . Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function . Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.

Mol Biol Cell, 2000 Feb, 11(2), 579 - 92
An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast; Luo W et al.; The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway . Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37 degrees C . Several vps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane . In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, and vps8 mutants . Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36 cells . After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane . In both vps1 and vps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface . Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1 . Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (and vps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64 . Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane . These data support a model in which mutant Pma1 is diverted from the Golgi to the surface in vps1 cells . We hypothesize that in vps8 and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.

Mol Biol Cell, 2000 Feb, 11(2), 543 - 54
The puc1 cyclin regulates the G1 phase of the fission yeast cell cycle in response to cell size; Martin-Castellanos C et al.; Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle . In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2 . Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive . At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC . Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1 . Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2 . However, when the puc1(+) gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells . This G1 delay is completely abolished in cells lacking rum1 . Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1.

Mol Biol Cell, 2000 Feb, 11(2), 435 - 52
Sbe2p and sbe22p, two homologous Golgi proteins involved in yeast cell wall formation; Santos B et al.; The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions . The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin . The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood . We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis . Although sbe2 and sbe22 null mutants are viable, sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure . First, sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37 degrees C and are hypersensitive to SDS and calcofluor . Second, electron microscopic analysis reveals that sbe2 sbe22 cells have an aberrant cell wall structure with a reduced mannoprotein layer . Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis . In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern . A Sbe2p-GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins . Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 and SBE22 . Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.

Mol Cell Biol, 2000 Mar, 20(6), 1947 - 55
The Est1 subunit of yeast telomerase binds the Tlc1 telomerase RNA; Zhou J et al.; Est1 is a component of yeast telomerase, and est1 mutants have senescence and telomere loss phenotypes . The exact function of Est1 is not known, and it is not homologous to components of other telomerases . We previously showed that Est1 protein coimmunoprecipitates with Tlc1 (the telomerase RNA) as well as with telomerase activity . Est1 has homology to Ebs1, an uncharacterized yeast open reading frame product, including homology to a putative RNA recognition motif (RRM) of Ebs1 . Deletion of EBS1 results in short telomeres . We created point mutations in a putative RRM of Est1 . One mutant was unable to complement either the senescence or the telomere loss phenotype of est1 mutants . Furthermore, the mutant protein no longer coprecipitated with the Tlc1 telomerase RNA . Mutants defective in the binding of Tlc1 RNA were nevertheless capable of binding single-stranded TG-rich DNA . Our data suggest that an important role of Est1 in the telomerase complex is to bind to the Tlc1 telomerase RNA via an RRM . Since Est1 can also bind telomeric DNA, Est1 may tether telomerase to the telomere.

RNA, 2000 Feb, 6(2), 206 - 19
A tertiary interaction detected in a human U2-U6 snRNA complex assembled in vitro resembles a genetically proven interaction in yeast; Valadkhan S et al.; U2 and U6 small nuclear RNAs are thought to play critical roles in pre-mRNA splicing catalysis . Genetic evidence suggests they form an extensively base-paired structure within the spliceosome that is required for catalysis . Especially in light of significant similarities with group II self-splicing introns, we wished to investigate whether the purified RNAs might by themselves be able to form a complex similar to that which appears to exist in the spliceosome . To this end, we synthesized and purified large segments of human U2 and U6 snRNAs . Upon annealing, the two RNAs efficiently formed a stable and apparently extensively base-paired (Tm = 50-60 degrees C in the presence of 20 mM Mg2+) complex . To investigate possible tertiary interactions, we subjected the annealed complex to UV irradiation, and two crosslinked species were identified and characterized . The major one links the second G in the highly conserved and critical ACAGAGA sequence in U6 with an A in U2 just 5' to U2-U6 helix Ia and opposite the invariant AGC in U6 . Remarkably, this crosslink indicates a tertiary interaction essentially identical to one detected previously by genetic covariation in yeast . Together our results suggest that purified U2 and U6 snRNAs can anneal and fold to form a structure resembling that likely to exist in the catalytically active spliceosome.

Plasmid, 2000 Mar, 43(2), 137 - 43
Telomere sequences attached to nuclearly migrated yeast linear plasmid; Takata H et al.; The yeast linear plasmid pCLU1, derived from pGKL1, has terminal proteins (TPs) covalently attached at the 5' ends of inverted terminal repeats (ITRs) and replicates in the cytoplasm, presumably using the TP as a primer for DNA synthesis . In Saccharomyces cerevisiae, under certain conditions, pCLU1 migrated into the nucleus and replicated in either linear or circular form . The linear-form plasmid lacked TPs; instead it carried host-telomere repeats at the ITR ends . The present study showed that (1) the added telomere was primarily composed of the repeated tracts of TGTGTGGGTGTGG, which was complementary to the RNA template of yeast telomerase, (2) the telomeric addition occurred at the very end of the ITRs, and (3) the sequence composition of the added telomeres was diverse among individual plasmids, but symmetrically identical at both ends of each plasmid . A similar mode of telomere addition was also observed in cells defective in the RAD52 gene .

Biochemistry, 2000 Feb 15, 39(6), 1475 - 88
Insulin-like signaling in yeast: modulation of protein phosphatase 2A, protein kinase A, cAMP-specific phosphodiesterase, and glycosyl-phosphatidylinositol-specific phospholipase C activities; Muller G et al.; Previously, we have described significant effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae under conditions of growth limitation . These regulations apparently rely on a transmembrane receptor capable of binding human insulin and responding by tyrosine/serine phosphorylation of a specific set of polypeptides {Muller, G., Rouveyre, N., Crecelius, A., and Bandlow, W . (1998) Biochemistry 37, 8683-8695; Muller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W . (1998) Biochemistry 37, 8696-8704; Muller, G., Rouveyre, N., Upshon, C., and Bandlow, W . (1998) Biochemistry 37, 8705-8713} . To characterize the molecular link between the initial steps in insulin-like signaling in yeast and the changes in the activities of glycogen synthase and glycogen phosphorylase, we examined here the effects of human insulin on a set of key regulatory enzymes of glycogen metabolism, protein phosphatase 2A (PP2A), cAMP-specific phosphodiesterase (cAMP-PDE), and protein kinase A (PKA) . PP2A was activated about 2-fold by insulin in spheroplasts and in intact cells, whereas the fraction of active PKA was significantly reduced in a cAMP-independent manner as well as through a subsequent up to 3-fold increase in particulate cAMP-PDE activity accompanied by a 50% decrease in cytosolic cAMP levels . In addition, glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC), which in isolated rat adipocytes is activated by insulin, was stimulated to up to 5-fold by glucose and 10-fold by glucose plus insulin in both yeast spheroplasts and intact cells leading to a concentration-dependent leftward shift of the glucose-response curve for activation of the GPI-PLC . GPI-PLC was most pronouncedly stimulated by authentic human insulin compared to various insulin analogues and insulin-like growth factor I . In addition to lipolytic cleavage by GPI-PLC, the GPI anchor of the cAMP-binding ectoprotein, Gce1p, was secondarily processed by a rapid proteolytic event . As the GPI-PLC reaction is rate limiting, the efficiency of the two-step anchor cleavage was significantly increased when insulin was present together with glucose as compared to glucose alone . The insulin concentrations effective in modulating PP2A, PKA, cAMP-PDE, and GPI-PLC activities correlate well with those required for half-saturation of the specific binding sites as well as for stimulation of protein phosphorylation and glycogen accumulation . The data suggest that mammalian insulin-sensitive cells and yeast share (part of) the key regulatory mechanism (consisting of PP2A, PKA, cAMP-PDE, and GPI-PLC) involved in the transduction of the insulin signal from the respective receptor systems to glycogen synthase and phosphorylase.

Nucleic Acids Res, 2000 Mar 15, 28(6), 1481 - 8
Analysis of the yeast transcriptome with structural and functional categories: characterizing highly expressed proteins; Jansen R et al.; We analyzed 10 genome expression data sets by large-scale cross-referencing against broad structural and functional categories . The data sets, generated by different techniques (e.g . SAGE and gene chips), provide various representations of the yeast transcriptome (the set of all yeast genes, weighted by transcript abundance) . Our analysis enabled us to determine features more prevalent in the transcriptome than the genome: i.e . those that are common to highly expressed proteins . Starting with simplest categories, we find that, relative to the genome, the transcriptome is enriched in Ala and Gly and depleted in Asn and very long proteins . We find, furthermore, that protein length and maximum expression level have a roughly inverse relationship . To relate expression level and protein structure, we assigned transmembrane helices and known folds (using PSI-blast) to each protein in the genome; this allowed us to determine that the transcriptome is enriched in mixed alpha-beta structures and depleted in membrane proteins relative to the genome . In particular, some enzymatic folds, such as the TIM barrel and the G3P dehydrogenase fold, are much more prevalent in the transcriptome than the genome, whereas others, such as the protein-kinase and leucine-zipper folds, are depleted . The TIM barrel, in fact, is overwhelmingly the 'top fold' in the transcriptome, while it only ranks fifth in the genome . The most highly enriched functional categories in the transcriptome (based on the MIPS system) are energy production and protein synthesis, while categories such as transcription, transport and signaling are depleted . Furthermore, for a given functional category, transcriptome enrichment varies quite substantially between the different expression data sets, with a variation an order of magnitude larger than for the other categories cross-referenced (e.g . amino acids) . One can readily see how the enrichment and depletion of the various functional categories relates directly to that of particular folds.

Nucleic Acids Res, 2000 Mar 15, 28(6), 1390 - 6
Different roles for abf1p and a T-rich promoter element in nucleosome organization of the yeast RPS28A gene; Lascaris RF et al.; In vivo mutational analysis of the yeast RPS28A ribosomal protein (rp-)gene promoter demonstrated that both the Abf1p binding site and the adjacent T-rich element are essential for efficient transcription . In vivo Mnase and DNaseI digestion showed that the RPS28A promoter contains a 50-60 bp long nucleosome-free region directly downstream from the Abf1p binding site, followed by an ordered array of nucleosomes . Mutating either the Abf1p binding site or the T-rich element has dramatic, but different, effects on the local chromatin structure . Failure to bind Abf1p appears to cause nucleosome positioning to become disorganized as concluded from the complete disappearance of Mnase hypersensitive sites . On the other hand, mutation of the T-rich element causes the downstream nucleosomal array to shift by approximately 50 bp towards the Abf1p site, resulting in loss of the nucleosome-free region downstream of Abf1p . We conclude that Abf1p is a strong organizer of local chromatin structure that appears to act as a nucleosomal boundary factor requiring the downstream T-rich element to create a nucleosome-free region.

Nucleic Acids Res, 2000 Mar 15, 28(6), 1313 - 21
Interaction of the yeast DExH-box RNA helicase prp22p with the 3' splice site during the second step of nuclear pre-mRNA splicing; McPheeters DS et al.; Using site-specific incorporation of the photo-chemical cross-linking reagent 4-thiouridine, we demonstrate the previously unknown association of two proteins with yeast 3' splice sites . One of these is an unidentified approximately 122 kDa protein that cross-links to 3' splice sites during formation of the pre--spliceosome . The other factor is the DExH-box RNA helicase, Prp22p . With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3' splice site is observed . In contrast, substrates blocked at the second step exhibit strong cross-linking of Prp22 to intron sequences at the 3' splice site, but not to adjacent exon sequences . In vitro reconstitution experiments also show that the association of Prp22p with intron sequences at the 3' splice site is dependent on Prp16p and does not persist when release of mature mRNA from the spliceosome is blocked . Taken together, these results suggest that the 3' splice site of yeast introns is contacted much earlier than previously envisioned by a protein of approximately 120 kDa, and that a transient association of Prp22p with the 3' splice site occurs between the first and second catalytic steps.

J Cell Biol, 2000 Feb 21, 148(4), 635 - 51
The yeast nuclear pore complex: composition, architecture, and transport mechanism; Rout MP et al.; An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine . Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins) . This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC . Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.

Biotechniques, 2000 Feb, 28(2), 328 - 30, 332-6
Approaches to detecting false positives in yeast two-hybrid systems; Serebriiskii I et al.; While many novel associations predicted by two-hybrid library screens reflect actual biological associations of two proteins in vivo, at times the functional co-relevance of two proteins scored as interacting in the two-hybrid system is unlikely . The reason for this positive score remains obscure, which leads to designating such clones as false positives . After investigating the effect of over-expressing a series of putative false positives in yeast, we determined that expression of some of these clones induces an array of biological effects in yeast, including altered growth rate and cell permeability, that bias perceived activity of LacZ reporters . Based on these observations, we identify four simple strategies that can assist in determining whether a protein is likely to have been selected in a two-hybrid screen because of indirect metabolic effects.

FEBS Lett, 2000 Feb 18, 468(1), 84 - 8
A conserved small GTP-binding protein Alp41 is essential for the cofactor-dependent biogenesis of microtubules in fission yeast; Radcliffe PA et al.; The proper folding of tubulins and their incorporation into microtubules consist of a series of reactions, in which evolutionarily conserved proteins, cofactors A to E, play a vital role . We have cloned a fission yeast gene (alp41(+)) which encodes a highly conserved small GTP-binding protein homologous to budding yeast CIN4 and human ARF-like Arl2 . alp41(+) is essential, disruption of which results in microtubule dysfunction and growth polarity defects . Genetic analysis indicates that Alp41 plays a crucial role in the cofactor-dependent pathway, in which it functions upstream of the cofactor D homologue Alp1(D) and possibly in concert with Alp21(E).

J Cell Sci, 2000 Mar, 113 ( Pt 6), 1075 - 88
A novel mutant allele of the chromatin-bound fission yeast checkpoint protein Rad17 separates the DNA structure checkpoints; Griffiths D et al.; To further dissect the genetic differences between the checkpoint pathway following S-phase cdc arrest versus DNA damage, a genetic screen was performed for checkpoint mutants that were unable to arrest mitosis following cell-cycle arrest with a temperature-sensitive DNA polymerase delta mutant, cdc20-M10 . One such checkpoint mutant, rad17-d14, was found to display the cut phenotype following S-phase arrest by cdc20-M10, but not by the DNA synthesis inhibitor hydroxyurea, reminiscent of the chk1 mutant . Unlike chk1 , rad17-d14 was not sensitive to UV irradiation . Interestingly, the ionising radiation sensitivity of rad17-d14 was only at higher doses, and cells were found to be defective in properly arresting cell division following irradiation in S phase, but not G(2) phase . Biochemical analysis attributes the checkpoint defects of rad17-d14 to the failure to phosphorylate the checkpoint effector Chk1p . To investigate if Rad17p monitors the genome for abnormal DNA structures specifically during DNA synthesis, chromatin association of Rad17p was analysed . Rad17p was found to be chromatin associated throughout the cell cycle, not just during S phase . This interaction occurred irrespective of the arrest with cdc20-M10 and, surprisingly, was also independent of the other checkpoint Rad proteins, and the cell-cycle effectors Chk1p and Cds1p.

J Bioenerg Biomembr, 1999 Dec, 31(6), 569 - 79
Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary molecular determinants; Azoulay-Zohar H et al.; The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wild type, porinless, or expressing recombinant human porin) was studied in an effort to identify minimal requirements for each component . A short hydrophobic N-terminal peptide of hexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria . Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loops around residues 110 and 200, as determined by proteolytic-fragment identification using antibodies . Recombinant human porin in yeast mitochondria is more sensitive to proteolysis than wild-type porin in rat liver mitochondria . Recombinant yeast mitochondria, harboring several natural or engineered porins from various sources, bind hexokinase to variable extent with marked preference for the mammalian porin1 isoform . Genetic alteration of this isoform at the C-, but not the N-terminal, results in a significant reduction of hexokinase binding ability . Macromolecular crowding (dextran) promotes a stronger association of the enzyme to all recombinant mitochondria, as well as to proteolytically digested organelles . Consequently, brain hexokinase association with heterologous mitochondria (yeast) in these conditions occurs to an extent comparable to that with homologous (rat) mitochondria . The study, also pertinent to the topology and organization of porin in the membrane, represents a necessary first step in the functional investigation of the physiological role of mammalian hexokinase binding to mitochondria in reconstituted heterologous recombinant systems, as models to cellular metabolism.

J Anim Sci, 2000 Jan, 78(1), 94 - 9
Efficacy of yeast phytase in improving phosphorus bioavailability in a corn-soybean meal-based diet for growing pigs; Matsui T et al.; Crossbred barrows (n = 66; 6 wk old) were used in a 6-wk experiment to evaluate the efficacy of phytase from yeast or Aspergillus niger on performance, tibial characteristics, and serum inorganic P concentration . We also investigated the stability of these phytases in acidic solutions with pepsin, which simulated gastric conditions . Pigs were fed a P-adequate diet containing .34% nonphytate-P or a low-P diet containing .20% nonphytate-P . The low-P diet was supplemented with 0, 1,000, 2,000, or 4,000 phytase units (PU; the activity at optimal pH, i.e., pH 4.2 for yeast phytase and pH 5.5 for phytase from Aspergillus niger)/kg of yeast phytase, or 1,000 PU/kg phytase from Aspergillus niger . The graded level of yeast phytase linearly increased ADG (P = .047), tibial weight (P = .091), tibial density (P < .001), and P concentration in tibial cortex (P = .018) . Aspergillus niger phytase also increased ADG (P = .022), serum inorganic P concentration (P < .001), tibial density (P = .007), and tibial P concentration (P = .025) . The pigs given 1,000 PU/kg Aspergillus niger phytase showed greater ADG (P = .091), tibial density (P= .001), and tibial P concentration (P = .062) than those given 1,000 PU/kg yeast phytase . No measurements differed (P > .31) between the pigs given 1,000 PU/kg Aspergillus niger phytase and those given 4,000 PU/kg yeast phytase . These results suggested that yeast phytase improves bioavailability of P in the diet for growing pigs but the efficacy of yeast phytase is less than that of Aspergillus niger phytase . During incubation in acidic solutions with pepsin, yeast phytase (P < .001) lost more of its activity than Aspergillus niger phytase . This lesser stability of yeast phytase may be responsible for the poorer efficacy of yeast phytase than that of Aspergillus niger . In summary, supplementation of swine diets with yeast phytase is beneficial, but its efficacy is less than that of Aspergillus niger phytase.

Wei Sheng Yan Jiu, 1998 Jul, 27(4), 257 - 8
{Study on the contamination level and the tolerable limit of mould and yeast in yoghurt}; Li F et al.; The results show that 67.33% of yoghurt samples collected from factories, supermarkets and retailers are contaminated by mould and yeast . The main problem of yoghurt product is yeast contamination (56.67%) . The highest count of mould and yeast in the positive samples are 39,500 cft/ml and innumerable, respectively . There is a significant difference in the levels of contamination of mould and yeast between yoghurt filled in the glass bottles and that in the plastic bottles (P < 0.005) . According to the results above, it is suggested that the tolerable limit of mould and yeast in yoghurt should be equal to or less than 50 cfu/ml.

J Biol Chem, 2000 Feb 25, 275(8), 5941 - 6
Subunit interactions in yeast transcription/repair factor TFIIH . Requirement for Tfb3 subunit in nucleotide excision repair; Feaver WJ et al.; A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro . Interestingly, tfb3(ts) failed to grow on medium containing caffeine . A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2 . These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome.

J Biol Chem, 2000 Feb 25, 275(8), 5845 - 51
The itinerary of a vesicle component, Aut7p/Cvt5p, terminates in the yeast vacuole via the autophagy/Cvt pathways; Huang WP et al.; Aminopeptidase I (API) is delivered to the yeast vacuole by one of two alternative pathways, cytoplasm to vacuole targeting (Cvt) or autophagy, depending on nutrient conditions . Genetic, morphological, and biochemical studies indicate that the two pathways share many of the same molecular components . The Cvt pathway functions during vegetative growth, while autophagy is induced during starvation . Both pathways involve the formation of cytosolic vesicles that fuse with the vacuole . In either case, the mechanism of vesicle formation is not known . Autophagic uptake displays a greater capacity for cytosolic protein sequestration . This suggests the involvement of an inducible protein(s) that allows the vesicle-forming machinery to adapt to the increased degradative needs of the cell . We have analyzed the biosynthesis of Aut7p, a protein required for both pathways . We find Aut7p expression is induced by nitrogen starvation . Aut7p is degraded by a process dependent on both proteinase A and Cvt/autophagy components . Protease accessibility assays demonstrate that Aut7p is located within vesicles in strains defective in vesicle delivery or breakdown . Finally, the aut7/cvt5 mutant accumulates precursor API at a stage prior to vesicle completion . These data suggest that Aut7p is induced during autophagy and delivered to the vacuole together with precursor API by Cvt/autophagic vesicles.

J Biol Chem, 2000 Feb 25, 275(8), 5573 - 81
The yeast STM1 gene encodes a purine motif triple helical DNA-binding protein; Nelson LD et al.; The formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination . Using conventional and affinity chromatography, we purified from Saccharomyces cerevisiae whole-cell extract a 35-kDa protein that avidly and specifically bound a purine motif triplex (with a K(d) of 61 pM) but not a pyrimidine motif triplex or duplex DNA . Peptide microsequencing identified this protein as the product of the STM1 gene . Confirmation that Stm1p is a purine motif triplex-binding protein was obtained by electrophoretic mobility shift assays using either bacterially expressed, recombinant Stm1p or whole-cell extracts from stm1Delta yeast . Stm1p has previously been identified as G4p2, a G-quartet nucleic acid-binding protein . This suggests that some proteins actually recognize features shared by G4 DNA and purine motif triplexes, e.g . Hoogsteen hydrogen-bonded guanines . Genetically, the STM1 gene has been identified as a multicopy suppressor of mutations in several genes involved in mitosis (e.g . TOM1, MPT5, and POP2) . A possible role for multiplex DNA and its binding proteins in mitosis is discussed.

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2134 - 9
Meiotic instability of CAG repeat tracts occurs by double-strand break repair in yeast; Jankowski C et al.; Expansion of trinucleotide repeats is associated with a growing number of human diseases . The mechanism and timing of expansion of the repeat tract are poorly understood . In humans, trinucleotide repeats show extreme meiotic instability, and expansion of the repeat tract has been suggested to occur in the germ-line mitotic divisions or postmeiotically during early divisions of the embryo . Studies in model organisms have indicated that polymerase slippage plays a major role in the repeat tract instability and meiotic instability is severalfold higher than the mitotic instability . We show here that meiotic instability of the CAG/CTG repeat tract in yeast is associated with double-strand break (DSB) formation within the repeated sequences, and that the DSB formation is dependent on the meiotic recombination machinery . The DSB repair results in both expansions and contractions of the CAG repeat tract.

Curr Opin Cell Biol, 2000 Feb, 12(1), 119 - 25
How centrioles work: lessons from green yeast; Marshall WF et al.; Centrioles are the organizing centers around which centrosomes assemble . Despite a century of study, the molecular details of centriole function and assembly remain largely unknown . Recent work has exploited the unique advantages of unicellular algae to reveal proteins that play central roles in centriole biology.

Curr Biol, 2000 Feb 10, 10(3), 145 - 8
Discrete in vivo roles for the MutL homologs Mlh2p and Mlh3p in the removal of frameshift intermediates in budding yeast; Harfe BD et al.; The DNA mismatch repair machinery is involved in the correction of a wide variety of mutational intermediates . In bacterial cells, homodimers of the MutS protein bind mismatches and MutL homodimers couple mismatch recognition to downstream processing steps {1} . Eukaryotes possess multiple MutS and MutL homologs that form discrete, heterodimeric complexes with specific mismatch recognition and repair properties . In yeast, there are six MutS (Msh1-6p) and four MutL (Mlh1-3p and Pms1p) family members {2} {3} . Heterodimers comprising Msh2p and Msh3p or Msh2p and Msh6p recognize mismatches in nuclear DNA {4} {5} and the subsequent processing steps most often involve a Mlh1p-Pms1P heterodimer {6} {7} . Mlh1p also forms heterodimeric complexes with Mlh2p and Mlh3p {8}, and a minor role for Mlh3p in nuclear mismatch repair has been reported {9} . No mismatch repair function has yet been assigned to the fourth yeast MutL homolog, Mlh2p, although mlh2 mutants exhibit weak resistance to some DNA damaging agents {10} . We have used two frameshift reversion assays to examine the roles of the yeast Mlh2 and Mlh3 proteins in vivo . This analysis demonstrates, for the first time, that yeast Mlh2p plays a role in the repair of mutational intermediates, and extends earlier results implicating Mlh3p in mismatch repair.

Gene, 2000 Feb 8, 243(1-2), 195 - 205
RLR1 (THO2), required for expressing lacZ fusions in yeast, is conserved from yeast to humans and is a suppressor of SIN4; West RW Jr et al.; We isolated a mutation (rlr1-1; required for lacZ RNA) in the Saccharomyces cerevisiae (Sc) RLR1 gene as a suppressor of sin4, a component of the Mediator subcomplex of the RNA polymerase II holoenzyme and a determinant of chromatin structure . RLR1 encodes a deduced protein found also in fission yeast, nematode worms, and humans . The presence of these orthologs suggests that Rlr1 family members comprise a class of putative KEKE motif-containing proteins, characteristic of certain chaperones as well as regulators and subunits of the mammalian 20S proteasome . A role for RLR1 (THO2) in transcription appears to occur at a step subsequent to transcription initiation (see also Piruat, J.I . and Aguilera, A., 1998 . EMBO J . 17, 4859-4872); Sc genes fused to the reporter gene lacZ were expressed at a very low level, while the corresponding native chromosomal genes were expressed at approximately normal levels in rlr1 mutants . Our studies show that rlr1 mutations cause a wide range of growth defects in addition to their novel affect on lacZ.

EMBO J, 2000 Feb 15, 19(4), 767 - 75
The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway; MacAlpine DM et al.; Mitochondrial DNA (mtDNA) is inherited as a protein-DNA complex (the nucleoid) . We show that activation of the general amino acid response pathway in rho(+) and rho(-) petite cells results in an increased number of nucleoids without an increase in mtDNA copy number . In rho(-) cells, activation of the general amino acid response pathway results in increased intramolecular recombination between tandemly repeated sequences of rho(-) mtDNA to produce small, circular oligomers that are packaged into individual nucleoids, resulting in an approximately 10-fold increase in nucleoid number . The parsing of mtDNA into nucleoids due to general amino acid control requires Ilv5p, a mitochondrial protein that also functions in branched chain amino acid biosynthesis, and one or more factors required for mtDNA recombination . Two additional proteins known to function in mtDNA recombination, Abf2p and Mgt1p, are also required for parsing mtDNA into a larger number of nucleoids, although expression of these proteins is not under general amino acid control . Increased nucleoid number leads to increased mtDNA transmission, suggesting a mechanism to enhance mtDNA inheritance under amino acid starvation conditions.

EMBO J, 2000 Feb 15, 19(4), 550 - 61
Degradation of unassembled Vph1p reveals novel aspects of the yeast ER quality control system; Hill K et al.; The endoplasmic reticulum quality control (ERQC) system retains and degrades soluble and membrane proteins that misfold or fail to assemble . Vph1p is the 100 kDa membrane subunit of the yeast Saccharomyces cerevisiae V-ATPase, which together with other subunits, assembles into the V-ATPase in the ER, requiring the ER resident protein Vma22p . In vma22Delta cells, Vph1p remains an integral membrane protein with wild-type topology in the ER membrane before undergoing a rapid and concerted degradation requiring neither vacuolar proteases nor transport to the Golgi . Failure to assemble targets Vph1p for degradation in a process involving ubiquitylation, the proteasome and cytosolic but not ER lumenal chaperones . Vph1p appears to possess the traits of a 'classical' ERQC substrate, yet novel characteristics are involved in its degradation: (i) UBC genes other than UBC6 and UBC7 are involved and (ii) components of the ERQC system identified to date (Der1p, Hrd1p/Der3p and Hrd3p) are not required . These data suggest that other ERQC components must exist to effect the degradation of Vph1p, perhaps comprising an alternative pathway.

FEBS Lett, 2000 Feb 11, 467(2-3), 337 - 40
Molecular cloning of Drosophila gamma-glutamylcysteine synthetase by functional complementation of a yeast mutant; Saunders RD et al.; gamma-Glutamylcysteine synthetase (GCS) catalyses a critical, rate-limiting step in glutathione synthesis . In this study we describe the isolation and characterisation of a GCS cDNA (pDmGCS4.3 . 3) from Drosophila melanogaster by functional complementation of a Saccharomyces cerevisiae gsh1 mutant . Expression of pDmGCS4.3.3 in the yeast mutant partially restored glutathione levels and conferred resistance to methylglyoxal . The pDmGCS4.3.3 cDNA was found to be approx . 4.6 kb in length, containing a 2 kb fragment encoding an open reading frame with a high degree of deduced amino acid sequence identity with previously reported GCS sequences . In situ hybridisation revealed that the Drosophila GCS gene maps to 7D6-9 on the X chromosome.

Gene, 2000 Jan 11, 241(2), 309 - 15
A highly representative two-hybrid genomic library for the yeast Yarrowia lipolytica; Kabani M et al.; Since its description by Fields and Song in 1989 (Nature 340, 245-246), the yeast two-hybrid system has been used extensively to study protein-protein interactions, becoming increasingly efficient with technological and methodological improvements . Here, we report the construction of a highly representative two-hybrid genomic library for the dimorphic yeast Yarrowia lipolytica based on the system described by James et al . (1996 . Genetics 144, 1425-1436) . The endoplasmic reticulum protein Slslp was then used as a bait in a functional test of the library . Indeed, we previously showed that the SLS1 gene product is involved in protein translocation across the endoplasmic reticulum membrane and interacts physically in a two-hybrid assay with Kar2p, an essential luminal member of the HSP70 family (Boisrame et al., 1998 . J . Biol . Chem . 273, 30 903-30 908) . We developed a mating strategy similar to that used for the Saccharomyces cerevisiae FRYL library (Fromont-Racine et al., 1997 . Nat . Genet . 16, 277-282) . No other partner than Kar2p was identified in this screen . As an interesting result, Kar2p interacts with Slslp through its ATPase domain, supporting our hypothesis that Slslp is a cofactor of the chaperone protein, modulating its activity during the HSP70 cycle . Our results indicate that we have constructed a new and powerful tool for the study of Yarrowia lipolytica, which we believe is a good alternative model to investigate such complex biological processes as secretion pathways.

Biochim Biophys Acta, 1999 Dec 23, 1489(2-3), 223 - 32
A second-site mutation at glutamate-257 that restores the function of the mutant yeast ribosomal protein L5 containing lysine-270,271-->arginine; Yeh LC et al.; A genetic approach was used to identify interacting regions of yeast ribosomal protein L5 (also known as L1, L1a, or YL3) . Previous studies from our laboratory showed that residues K270 and K271 in protein L5 are essential for its function . The mutant L5 protein in which both residues were replaced by arginine residues (K270,271R) exhibited about 80% RNA binding capability compared to the wild-type and the mutant protein was assembled into the 60S ribosomal subunits in vivo . The yeast strain expressing this mutant protein in a homozygous form was lethal (Biochim . Biophys . Acta 1308 (1996) 133-141) . In the present study, this non-functional mutant was used to select intragenic suppressors . A spontaneous, intragenic suppressor which contained an E257K substitution (in addition to the primary mutations) was identified . The suppressor protein bound about 60% of yeast 5S rRNA in vitro compared to the wild-type . To gain more insight into the nature of the intragenic suppressor, additional mutant proteins in which E257 was substituted by a variety of amino acids were produced by site-directed mutagenesis . The ability of each mutant protein to bind yeast 5S rRNA in vitro and to suppress the lethal effect of the double K270,271 mutation in vivo were examined . Results suggest communication between two non-contiguous domains on protein L5 and that several factors, such as electrostatic interaction and hydrogen bonding are likely to play a role in this global communication . Mutation studies on E257 alone also reveal that substitutions of this residue in L5 protein could affect cell growth under specified conditions, but a variety of changes could be tolerated without serious deleterious effects . We propose a working model in which E257 is located in a loop and the dynamic as well as the flexibility of this loop is important for L5 function.

Mol Microbiol, 2000 Feb, 35(3), 553 - 65
Differential post-transcriptional regulation of yeast mRNAs in response to high and low glucose concentrations; Yin Z et al.; Glucose regulates yeast gene expression at both transcriptional and post-transcriptional levels . Glucose strongly represses the transcription of the gluconeogenic genes, FBP1 and PCK1, and accelerates the degradation of their mRNAs . Together these mechanisms are responsible for the rapid decrease in gluconeogenic enzyme synthesis when yeast cells switch to glycolytic metabolism . In this study, we show that accelerated gluconeogenic mRNA degradation can be triggered by low concentrations of glucose (<0 . 02%) . This sets the FBP1 and PCK1 mRNAs apart from other glucose-sensitive mRNAs, such as the Ip mRNA, which only responds to high glucose concentrations (>1%) . We also show that accelerated gluconeogenic mRNA degradation is co-ordinated with transcriptional repression by common signalling components that include sugar kinases and Ras-cAMP signalling . Furthermore, the ability of the low glucose signal to trigger accelerated gluconeogenic mRNA degradation depends upon the low glucose sensor, Snf3p, but not on the high glucose sensor, Rgt2p . Also, this response is influenced by reg1 and ume5 mutations, but not by grr1 or rgt1 mutations . Our data suggest that several signalling pathways co-ordinate differential post-transcriptional and transcriptional responses in yeast, depending upon the amount of glucose available in the medium.

J Biol Chem, 2000 Feb 18, 275(7), 4635 - 9
N(alpha)-acetylation and proteolytic activity of the yeast 20 S proteasome; Kimura Y et al.; N(alpha)-acetylation, catalyzed co-translationally with N(alpha)-acetyltransferase (NAT), is the most common modifications of eukaryotic proteins . In yeast, there are at least three NATs: NAT1, MAK3, and NAT3 . The 20 S proteasome subunits were purified from the normal strain and each of the deletion mutants, nat1, mak3, and nat3 . The electrophoretic mobility of these subunits was compared by two-dimensional gel electrophoresis . Shifts toward the alkaline side of the gel and unblocking of the N terminus of certain of the subunits in one or another of the mutants indicated that the alpha1, alpha2, alpha3, alpha4, alpha7, and beta3 subunits were acetylated with NAT1, the alpha5 and alpha6 subunits were acetylated with MAK3, and the beta4 subunit was acetylated with NAT3 . Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the alpha5 and alpha6 subunits, respectively, extended the known types of MAK3 substrates . Thus, nine subunits were N (alpha)-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region . The 20 S proteasomes derived from either the nat1 mutant or the normal strain were similar in respect to chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities in vitro, suggesting that N(alpha)-acetylation does not play a major functional role in these activities . However, the chymotrypsin-like activity in the absence of sodium dodecyl sulfate was slightly higher in the nat1 mutant than in the normal strain.

J Microbiol Methods, 2000 Feb, 39(3), 253 - 64
Assessment of the intracellular pH of immobilized and continuously perfused yeast cells employing fluorescence ratio imaging analysis; Breeuwer P et al.; The intracellular pH (pHin) of Saccharomyces cerevisiae was measured employing fluorescence ratio imaging microscopy (FRIM) . The yeast cells were fluorescently labeled with the pH dependent probe 5(and-6)-carboxyfluorescein (cF) or 5(and-6)-carboxyfluorescein succinimidyl ester (cFSE), and subsequently attached to ferric nitrate pretreated glass slides . The labeled and adhered cells could still divide and were metabolically active . Measurement of the pHin was performed during continuous perfusion of the cells with buffer or medium . Cells labeled with cF are highly fluorescent and in non-energized cells the pHin could be easily measured . However, in energized yeast cells cF was accumulated in the vacuoles and/or exported to the extracellular environment, most likely by an energy-dependent transport system, thus limiting the time period over which the pHin can be effectively measured . Therefore, cFSE (which conjugates with aliphatic amines in the cytoplasm) was applied to prevent translocation of fluorescent probe to the vacuole and/or extracellular environment . The continuous perfusion in combination with the cFSE labeling of the immobilized cells was successfully applied to determine the effect of low and high pHin and addition of glucose on the pHin of individual yeast cells over a long time period.

Fungal Genet Biol, 1999 Dec, 28(3), 227 - 37
The frost gene of Neurospora crassa is a homolog of yeast cdc1 and affects hyphal branching via manganese homeostasis; Sone T et al.; The Neurospora crassa mutant frost has a hyperbranching phenotype that can be corrected by adding Ca(2+), suggesting that characterization of this gene might clarify the mechanism of Ca(2+)-dependent tip growth . The wild-type allele was cloned by sib selection using protoplasts from arthroconidia . RFLP analysis revealed that the cloned DNA fragment mapped to the fr locus . The nucleotide sequence of genomic and cDNA was determined . The deduced amino acid sequence showed homology to the Saccharomyces cerevisiae CDC1 protein, implicated in manganese homeostasis . The fr mutant was sensitive to Mn(2+), and a revertant allele whose product differs by one amino acid was tolerant to Mn(2+) . Mn(2+) depletion induced the wild-type strain to hyperbranch, resulting in a morphology similar to that of fr . The fr mutant was also sensitive to calcineurin inhibitors . These results suggest that fr is involved in Mn(2+) homeostasis and point to a role for Mn(2+) in Neurospora branching .

Diagn Microbiol Infect Dis, 1999 Dec, 35(4), 271 - 3
Evaluation of the RapID Yeast Plus System for the identification of yeast; Moghaddas J et al.; We evaluated the RapID Yeast Plus System using 117 fresh and frozen clinical yeast isolates . The Uni-Yeast-Tek System was used to establish the correct identification . The Vitek System was used as the arbiter for any discrepant results, along with morphology . Of 117 isolates tested, the RapID Yeast Plus System identified 96.6% correctly . The RapID Yeast Plus System is an accurate and reliable alternative to other commonly used yeast identification systems.

Acta Crystallogr D Biol Crystallogr, 1999 Dec, 55 ( Pt 12), 2047 - 8
Crystallization and preliminary crystal analysis of yeast hexokinase PI and PII; Kuser PR et al.; Hexokinase is the prime enzyme of the Embden-Meyerhof pathway and is responsible for the first stage of energy conversion . It catalyzes the transfer of a phosphate to glucose to form glucose-6-phosphate . Yeast hexokinase PII is also known to play an important role in glucose signal transduction . Crystals of yeast hexokinase isoforms PI and PII were obtained by vapour-diffusion techniques using the hanging-drop method . Isoform PI crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 62.12, b = 78.87, c = 144.74 A . Unit-cell parameters for isoform PII crystals are a = b = 142.81, c = 58.46 A and the space group is I4 . Synchrotron diffraction data have been collected to 2.2 A resolution from the isoform PII crystal, whereas isoform PI diffracted to 3.1 A.

Exp Cell Res, 2000 Feb 25, 255(1), 95 - 101
Telomere shortening by cisplatin in yeast nucleotide excision repair mutant; Ishii K et al.; Telomeres are unique DNA tandem repeats that form the ends of eukaryotic chromosomes to protect the chromosomes from degradation and illegitimate recombination . In yeast, loss of telomere may be compensated for through the acquisition of new telomere by RAD52-mediated or RAD52-independent recombinational repair . In this report, the effects of cis-dichlorodiammine-platinum (II) (cisplatin) on telomere length and the role of nucleotide excision repair in telomere maintenance were examined in the yeast Saccharomyces cerevisiae . We showed that the SSL2 (RAD25) DNA repair yeast mutant exhibited a gradual shortening of the telomere in the presence of cisplatin . Further telomere shortening was prevented upon the withdrawal of cisplatin . Complementation of the mutant with the wild-type SSL2 (RAD25) gene abolished the cisplatin-induced telomere degradation . These results suggest that telomeres are susceptible to cisplatin-induced intrastrand crosslinks and that Ssl2 (Rad25) or the nucleotide excision repair pathway may play a critical role in the repair and the maintenance of telomere integrity .

FEBS Lett, 2000 Feb 4, 467(1), 22 - 6
The Leu-132 of the Ste4(Gbeta) subunit is essential for proper coupling of the G protein with the Ste2 alpha factor receptor during the mating pheromone response in yeast; Ongay-Larios L et al.; In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response . We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered alpha pheromone response in yeast cells . Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine . This residue lies in the first blade of the beta propeller structure proposed by crystallographic analysis . By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains . When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and alpha cells . The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor . These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gbeta and the Galpha subunits.

Curr Biol, 2000 Jan 27, 10(2), 111 - 4
Two paralogs involved in transcriptional silencing that antagonistically control yeast life span; Roy N et al.; In the yeast Saccharomyces cerevisiae, one determinant of aging or life span is the accumulation of extrachromosomal copies of rDNA circles in old mother cells {1} . The production of rDNA circles depends upon intrachromosomal recombination within the rDNA tandem array, a process regulated by the protein Sir2 (Sir2p) . Together with Sir1p, Sir3p, Sir4p and Orc1p, Sir2p is also involved in transcriptional silencing of genes at the silent mating-type cassettes, in the rDNA array, and at telomeres . Using a 'triple silencer' strain that can monitor an increase or decrease in gene expression at these three loci, we found that deletion of the ZDS1 gene caused an increase in silencing in the rDNA and at a silent mating-type cassette at the expense of telomere silencing . The zds1 deletion also resulted in an increase in life span and a decrease in Sir3p phosphorylation . In contrast, deletion of its paralog ZDS2 caused a decrease in rDNA silencing, a decrease in life span and an increase in Sir3p phosphorylation . As Zds2p, but not Zds1p, had strong two-hybrid interactions with Orc1p and the four Sir proteins, Zds1p might indirectly control Sir3p through a Sir3p kinase.

Mol Cell Biol Res Commun, 1999 Sep-Dec, 2(3), 178 - 84
The budding yeast Cdc15 localizes to the spindle pole body in a cell-cycle-dependent manner; Cenamor R et al.; Exit from mitosis in the budding yeast Saccharomyces cerevisiae cell cycle is regulated by a regulatory network that involves, among other proteins, the small GTPase Tem1, the protein phosphatase Cdc14, and the protein kinases Dbf2 and Cdc15 . Using a fusion to jellyfish green fluorescent protein (GFP), here we report that Cdc15 costains with the microtubular-organizing apparatus and that this localization is precluded in a mutant lacking the outer plaque of the spindle pole body (SPB) . The appearance of Cdc15 in the SPB is asymmetric and cell-cycle-regulated, preferentially marking the daughter cell SPB at anaphase and eventually disappearing at cytokinesis . Overproduction of GFP-tagged Cdc15 led to an accumulation of the fusion protein in both mother and daughter cells SPBs and, transiently, in small budded cells and shmoos . The Cdc15 localization pattern was maintained in dbf2, cdc14 and anaphase-promoting complex (cdc16) mutants, suggesting that the function of these proteins is not related to the localization of Cdc15 to the SPB but rather, at least in the case of Cdc14, to its timely removal from this structure . Tem1-depleted cells kept alive by Cdc15-GFP overexpression still display a proper localization of Cdc15 . The results presented here suggest that the transient cell-cycle-dependent localization of Cdc15 to the SPB plays a role in the regulation of the latest stages of the cell cycle.

Biotechnol Prog, 2000 Jan-Feb, 16(1), 31 - 7
Fine affinity discrimination by yeast surface display and flow cytometry; VanAntwerp JJ et al.; Yeast surface display is a eucaryotic system for the directed evolution of protein binding and stability . For antibody affinity maturation, achievable single-pass enrichment factors are a critical variable . Both reliable recovery of rare clones (yield) and effective differentiation between clones of only slightly improved affinity (purity) are paramount . To validate yeast display's purification potential, trial sorting experiments were performed . The D1.3 (anti-hen egg lysozyme) single chain variable fragment antibody and a 2-fold higher affinity mutant (M3) were each displayed on the surface of Saccharomyces cerevisiae . M3-displaying cells were mixed into the D1.3-displaying cells at a ratio of 1:1000 . Cells were fluorescently labeled according to antigen equilibrium binding and then sorted using a flow cytometer . Single-pass enrichment of M3-displaying cells was 125-fold (+/- 65-fold) . This level of performance is achievable because of the precision and reproducibility of optimal labeling conditions . This work further demonstrates the capability of yeast display for very fine discrimination between mutant clones of similar affinity . Because large improvements in affinity typically result from combinations of small changes, this capability to identify subtle improvements is essential for rapid affinity maturation.

J Cell Biol, 2000 Feb 7, 148(3), 441 - 52
Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast; Segal M et al.; The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions . In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud . Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells . As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex . Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck . Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell . Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity . In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

Membr Cell Biol, 1999, 13(1), 59 - 67
Different modes of ozone-induced lipid oxidation in Candida utilis yeast cells and isolated membrane preparations; Matus VK et al.; Significant differences in the development of ozonolysis of lipids in membrane preparations and intact cells of the Candida utilis yeast were revealed . First, unlike isolated membranes, in which lipid modifications can be initiated by low ozone doses (< 0.5 micromol O3/mg protein) and develop proportionally to the treatment dose, in intact yeast cells, even the most ozone-sensitive sterols and nitrogen-containing phospholipids (phosphatidylcholine and phosphatidylethanolamine) did not undergo oxidative destruction at doses up to 6.0 micromol O3/mg protein . Second, the peculiarity of the ozone-initiated lipid modification in intact cells was that different classes of lipids exhibited different sensitivity to ozone . With an increase in the ozone dose, neutral lipids (sterols) and nitrogen-containing phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) were modified to a greater extent . Third, the accumulation of lipid peroxidation products upon ozone treatment of cells, in contrast to the isolated membranes, was absent at low ozone doses and was recorded only after the lethal damage . It is suggested that these differences are related to both the function of antioxidative enzymes (catalase, superoxide dismutase, peroxidase, etc.) and the difference between the structural states (i.e., stability and accessibility to oxidation) of lipids in the isolated membranes and the intact cells.

J Biol Chem, 2000 Feb 11, 275(6), 4422 - 8
Three aromatic amino acid residues critical for galactose transport in yeast Gal2 transporter; Kasahara T et al.; Tyr(446) in putative transmembrane segment 10 (TM10) of the yeast galactose transporter Gal2 has previously been identified as essential for galactose recognition . In the present study, alignment of the amino acid sequences of 63 sugar transporters or related proteins revealed 14 aromatic sites, including Tyr(446) of Gal2, that are conserved in >75% of these proteins . The importance of the remaining 13 conserved aromatic amino acids was examined individually by random mutagenesis using degenerate primers . Galactose transport-positive clones were identified by plate selection and subjected to DNA sequencing . For those transport-positive clones corresponding to Tyr(352), and Phe(504) mutants, all the amino acid substitutions comprised aromatic residues . The importance of the aromatic residues at these sites was further investigated by replacing them individually with each of the other 19 amino acids and measuring the galactose transport activity of the resulting mutants . Among both Tyr(352) and Phe(504) mutants, the other aromatic amino acids supported galactose transport; no other amino acids conferred high affinity transport activity . Thus, at least three aromatic sites are critical for galactose transport: one at the extracellular boundary of putative TM7 (Tyr(352)), one in the middle of putative TM10 (Tyr(446)), and one in the middle of putative TM12 (Phe(504)).

Mol Gen Genet, 2000 Jan, 262(6), 1103 - 12
Rna14p, a component of the yeast nuclear cleavage/polyadenylation factor I, is also localised in mitochondria; Rouillard JM et al.; RNA14 was identified as a gene involved in premessenger RNA cleavage and polyadenylation . These processing steps take place in the nucleus, but the Rna14p protein is distributed in both the nucleus and the cytoplasm . By subcellular fractionation, we show here that the cytoplasmic fraction is localised in the mitochondria . In order to understand the role played by Rna14p in mitochondria, we have searched for new thermosensitive alleles of RNA14 . We isolated thirteen new mutants . Some of them are deficient in mRNA cleavage and polyadenylation at the restrictive temperature - like the first mutant identified (rna14-1) . However, others do not appear to be impaired in any of the steps in RNA metabolism investigated, nor do they appear to be involved in the replication or expression of mitochondrial DNA or in respiration . The localisation data strongly suggest that, besides an essential function in mRNA polyadenylation, the Rna14p protein has a non essential function in mitochondrial metabolism.

Mol Gen Genet, 2000 Jan, 262(6), 940 - 8
Extragenic suppressors that rescue defects in the heat stress response of the budding yeast mutant tom1; Sasaki T et al.; The TOM1 gene codes for a so-called HECT protein, a putative ubiquitin ligase, in Saccharomyces cerevisiae . Deletion of the entire gene (tom1-10) or the sequence encoding the HECT domain (tom1-2) causes temperature sensitivity for growth . Here we report the isolation of extragenic, recessive suppressors of tom1-2, which were designated tmr (for tom1 revertant) mutations . These were classified into eight complementation groups and six of the genes were identified: tmr1/cyr1, tmnr2/sch9, tmr3/zuo1, tmr4, tmr5/mot1, tmr6/sse1, tmr7 and tmr8/kre6 . These results suggested that the tom1 phenotype can be rescued by down-regulating the cAMP/PKA pathway . It was found that the temperature sensitivity of the tom1-2 mutant is indeed suppressed by multiple copies of PDE2 or BCY1, which encode negative regulators of the cAMP/PKA pathway . The MSN2 gene, which encodes a zinc-finger transcription factor involved in the general stress response is also a multicopy suppressor of tom1 . It was found that induction levels of both STRE-mediated (general stress response) and HSE-mediated gene expression (heat shock response) upon shift to high temperature are reduced by more than half in the tom1 mutant . Most of the isolated tmr mutations rescued one of the defects seen in both types of heat stress response in the tom1 mutant.

Cell, 2000 Jan 21, 100(2), 277 - 88
Molecular basis of a yeast prion species barrier; Santoso A et al.; The yeast {PSI+} factor is inherited by a prion mechanism involving self-propagating Sup35p aggregates . We find that Sup35p prion function is conserved among distantly related yeasts . As with mammalian prions, a species barrier inhibits prion induction between Sup35p from different yeast species . This barrier is faithfully reproduced in vitro where, remarkably, ongoing polymerization of one Sup35p species does not affect conversion of another . Chimeric analysis identifies a short domain sufficient to allow foreign Sup35p to cross this barrier . These observations argue that the species barrier results from specificity in the growing aggregate, mediated by a well-defined epitope on the amyloid surface and, together with our identification of a novel yeast prion domain, show that multiple prion-based heritable states can propagate independently within one cell.

Cell Motil Cytoskeleton, 2000 Feb, 45(2), 106 - 20
Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly; Freeman NL et al.; Control of cell shape and motility requires rearrangements of the actin cytoskeleton . One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56) . CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling . In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity . CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function . We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton . CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types . To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein . We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers . Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells . Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton .

Environ Health Perspect, 2000 Feb, 108(2), 97 - 103
Environmental estrogens induce transcriptionally active estrogen receptor dimers in yeast: activity potentiated by the coactivator RIP140; Sheeler CQ et al.; We used three yeast genetic systems to investigate the estrogen-like activity of octylphenol (OP), bisphenol-A (BPA), o,p'-DDT, and o, p'-DDE to induce human estrogen receptor (hER) dimerization and transcriptional activation . We have demonstrated that OP, BPA, and o, p'-DDT can induce hER ligand-dependent dimerization using a yeast two-hybrid assay . All three xenoestrogens, plus estradiol, enhanced estrogen response element (ERE)-dependent transcriptional activation of hER . In the presence of receptor interacting protein 140 (RIP140), ERE-dependent activity was dramatically amplified by 100-fold for estradiol, OP, BPA, and o,p'-DDT . A yeast whole-cell {(3)H}estradiol binding assay was developed to determine the site of interaction on the hER . We determined nonspecific binding by parallel incubations run in the presence of 5 microM unlabelled estradiol in PCY2 yeast . At the concentrations tested, unlabeled estradiol, OP, and BPA displaced {(3)H}estradiol in this binding assay, whereas the concentrations of o,p'-DDT and o,p'-DDE tested were insufficient to inhibit binding . Incubating yeast in the presence of increasing concentrations of estradiol and OP (1 microM) or BPA (1 microM) neither blocked nor altered the effect of estradiol on hER activity . We observed no agonistic activity of o,p'-DDE in any of the yeast models used . These results suggest that OP, BPA, and o,p'-DDT exert their estrogen-like activity through the ER in a manner similar to that of estradiol, and the coactivator RIP140 markedly potentiates this activity.

Proteins, 2000 Feb 1, 38(2), 226 - 38
Role of hydrophobic interactions in yeast phosphoglycerate kinase stability; Receveur V et al.; Cold denaturation of yeast phosphoglycerate kinase (yPGK) was investigated by a combination of far UV circular dichroism (CD), steady-state and time-resolved fluorescence, and small angle X-ray scattering . It was shown that cold denaturation of yPGK cannot be accounted for by a simple two-state process and that an intermediate state can be stabilized under mild denaturing conditions . Comparison between far UV CD and fluorescence shows that in this state the protein displays a fluorescence signal corresponding mainly to exposed tryptophans, whereas its CD signal is only partially modified . Comparison with spectroscopic data obtained from a mutant missing the last 12 amino-acids (yPGK delta404) suggests that lowering the temperature mainly results in a destabilization of hydrophobic interactions between the two domains . Small angle X-ray scattering measurements give further information about this stabilized intermediate . At 4 degrees C and in the presence of 0.45 M Gdn-HCl, the main species corresponds to a protein as compact as native yPGK, whereas a significant proportion of ellipticity has been lost . Although various techniques have shown the existence of residual structures in denatured proteins, this is one example of a compact denatured state devoid of its main content in alpha helices.

Nat Cell Biol, 2000 Feb, 2(2), 117 - 24
Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating; Shimada Y et al.; Cytoskeletal rearrangements during the cell cycle and in response to signals are regulated by small Rho-type GTPases, but it is not known how these GTPases are activated in a spatial and temporal manner . Here we show that Cdc24, the guanine-nucleotide exchange factor for the yeast GTPase Cdc42, is sequestered in the cell nucleus by Far1 . Export of Cdc24 to a site of cell polarization is mediated by two mechanisms . At bud emergence, activation of the G1 cyclin-dependent kinase Cdc28-Cln triggers degradation of Far1 and, as a result, relocation of Cdc24 to the cytoplasm . Cells overexpressing a non-degradable Far1 were unable to polarize their actin cytoskeleton because they failed to relocate Cdc24 to the incipient bud site . In contrast, in response to mating pheromones, the Far1-Cdc24 complex is exported from the nucleus by Msn5 . This mechanism ensures that Cdc24 is targeted to the site of receptor-associated heterotrimeric G-protein activation at the plasma membrane, thereby allowing polarization of the actin cytoskeleton along the morphogenetic gradient of pheromone . Either degradation of Far1 or its nuclear export by Msn5 was sufficient for cell growth, suggesting that the two mechanisms are redundant for cell viability . Taken together, our results indicate that Far1 functions as a nuclear anchor for Cdc24 . This sequestration regulates cell polarity in response to pheromones by restricting activation of Cdc42 to the site of pheromone receptor activation.

Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1143 - 7
Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins; Ito T et al.; Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell . As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae . We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MATalpha strain, and subsequently divided them into pools, each containing 96 clones . These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging . Our initial examination of approximately 4 x 10(6) different combinations, constituting approximately 10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel . Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport . The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.

Genetics, 2000 Feb, 154(2), 557 - 71
A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II; Conrad NK et al.; Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) in pre-mRNA processing . The yeast NRD1 gene encodes an essential RNA-binding protein that shares homology with mammalian CTD-binding proteins and is thought to regulate mRNA abundance by binding to a specific cis-acting element . The present work demonstrates genetic and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase CTDK-I . Previous studies have shown that Nrd1p associates with the CTD of pol II in yeast two-hybrid assays via its CTD-interaction domain (CID) . We show that nrd1 temperature-sensitive alleles are synthetically lethal with truncation of the CTD to 9 or 10 repeats . Nab3p, a yeast hnRNP, is a high-copy suppressor of some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast two-hybrid assay, and coimmunoprecipitates with Nrd1p . Temperature-sensitive alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown to phosphorylate the CTD and increase elongation efficiency in vitro . This set of genetic and physical interactions suggests a role for yeast RNA-binding proteins in transcriptional regulation.

Curr Genet, 1999 Dec, 36(6), 329 - 38
The budding yeast cohesin gene SCC1/MCD1/RHC21 genetically interacts with PKA, CDK and APC; Heo SJ et al.; Cohesin is a protein that plays a key role in the cohesion and separation of sister chromatids . During the duplication of chromatids, cohesin holds sister chromatids together until the onset of anaphase, and thereby prevents the premature separation of sister chromatids which would otherwise jeopardize the faithful segregation of chromosomes . To investigate the molecular mechanisms of sister chromatid cohesion, we have isolated multicopy suppressors of a temperature-sensitive (ts) mutation in the SCC1/MCD1/RHC21 gene which encodes a component of the cohesin complex in budding yeast . Isolation of multicopy suppressors of rhc21-sk16 and further genetic analyses revealed that several distinct biological pathways are involved in the regulation of SCC1/MCD1/RHC21 function . Firstly, PDE2 and BCY1, each of which inhibits the activity of protein kinase A (PKA), suppressed the temperature sensitivity of the rhc21-sk16 mutant . Secondly, PDE2 suppressed the temperature sensitivity of the cdc16-1 mutant . These results suggest that SCC1/MCD1/RHC21 is negatively regulated by the PKA pathway via the anaphase promoting complex (APC) . Thirdly, ZDS1, a multicopy suppressor of cdc28-1N, and its homologue ZDS2 were isolated as multicopy suppressors of rhc21-sk16 . Furthermore, the rhc21-sk16 mutant did not grow in the presence of the cdc28-1N mutation . Hence, SCC1/MCD1/RHC21 is positively regulated by the mitotic CDK, CDC28 . Finally, SCC1/MCD1/RHC21 was found to interact genetically with CDC20, an activator of APC . Overexpression of CDC20 suppressed the temperature sensitivity of rhc21-sk16, and rhc21-sk16 was shown to be synthetically lethal with cdc20-1 . In addition, the growth of the rhc21-sk16 mutant was inhibited by overproduction of the anaphase inhibitor Pds1p, whose degradation is mediated by Cdc20p in APC-dependent proteolysis . The functional relationships between SCC1/MCD1/RHC21 and PKA, CDK or APC are discussed.

Pflugers Arch, 2000, 439(3 Suppl), R73 - 5
Effect of cultivation mode on a bioprocess for chromium yeast biomass enrichment; Batic M et al.; Defined cultivation media for yeast growth which contained 278.8 mM of glucose and 0.1 mM of chromium(III) added as K2Cr(SO4)2 x 12 H2O was used in batch and combined batch/fed-batch cultivation mode . In fed batch cultivation mode the rate of substrate addition remained constant during growth of yeast and corresponded to a growth rate of 0.25 h(-1) . In both cases the growth and yeast activity was followed by on line measurement of optical density, pH and pO2 at 30 degrees C . At the end of the bioprocess the concentration of protein in yeast biomass was determined off line by the biuret reaction . Total and organically bound chromium was detected by ETA-AAS . Different cultivation modes affected the total cell protein concentration of yeast grown in media supplemented with chromium . In batch process the protein content represented 25.7% of dry yeast biomass, in contrast in the mixed bioprocess this value was 16.9% one the same period of time . The influence of cultivation mode on chromium uptake was seen in total chromium accumulation which reached 8.68 +/- 0.16 micromol g(-1) d.wt . in batch and 1.92 +/- 0.04 micromol of chromium g(-1) of dry yeast biomass in combined batch/fed-batch cultivation mode . The opposite was observed for organically bound chromium . The 60% of total accumulated chromium was organically bound during yeast growth in combined batch/fed-batch mode . When yeast was grown in batch mode this value attained 13.5% . Results suggested that a combined batch/fed-batch mode of cultivation was more effective over a batch system in chromium biotransformation to organically bound chromium, regardless of the lower protein ratio determined in the yeast biomass.

Trends Genet, 2000 Feb, 16(2), 51 - 3
RAP, RAP, open up! New wrinkles for RAP1 in yeast; Morse RH; RAP1 (repressor/activator protein 1) from budding yeast is well known for its involvement in gene activation and repression, telomere structure and function, and replication . Recent studies have examined additional roles for RAP1 in heterochromatin boundary-element formation, creation of hotspots for meiotic recombination, and chromatin opening . These studies provide new insight into the ability of this abundant DNA-binding protein to participate in a diverse array of functions taking place in a chromatin environment.

Trends Genet, 2000 Feb, 16(2), 49 - 51
The awesome power of yeast biochemical genomics; Carlson M; A new genomic strategy for identifying the gene encoding any biochemical activity has recently been developed, in which an array of individual yeast strains expressing a genomic set of open reading frames fused to glutathione S-transferase can be assayed for a biochemical activity of interest . Designated 'biochemical genomics', this approach represents an innovative application of genomic information.

J Biol Chem, 2000 Feb 4, 275(5), 3455 - 61
Peroxisomal membrane protein Pmp47 is essential in the metabolism of middle-chain fatty acid in yeast peroxisomes and Is associated with peroxisome proliferation; Nakagawa T et al.; Pmp47 of the methylotrophic yeast Candida boidinii belongs to a mitochondrial family of solute transporters and is localized in peroxisomal membranes . Its human homolog, Pmp34, is also known . In this study, we characterized the role of Pmp47 in fatty acid metabolism and peroxisome proliferation using the PMP47-deleted strain of C . boidinii (strain pmp47Delta) . The wild-type strain grew well on a middle-chain fatty acid, laureate, as the single carbon source, and mild peroxisome proliferation was observed during its growth . The pmp47Delta strain could not grow on laureate but could grow on long-chain fatty acids including palmitate, myristate, and oleate . The levels of laureate oxidation activity in intact cells and in semi-permeabilized cells of strain pmp47Delta were lower than the respective level in the wild-type strain, although the level of laureate oxidation activity in the cell lysate and the level of lauroyl-CoA oxidation in semi-permeabilized cells of strain pmp47Delta were indistinguishable from the respective level in the wild-type strain . When lauroyl-CoA was provided in the cytosol of strain pmp47Delta through expression of Saccharomyces cerevisiae Faa2p (lauroyl-CoA synthetase) in which its peroxisome targeting signal was deleted, the growth of strain pmp47Delta on laureate was recovered to the level of growth of the wild-type strain . Laureate is converted to its CoA form in peroxisomes by the action of lauroyl-CoA synthetase . These results suggested that Pmp47 is involved in the transport of a small molecule (possibly ATP) required in the conversion of laureate to its CoA form in peroxisomes and that the absence of Pmp47 causes impairment of laureate metabolism, which results in the inability of pmp47Delta cells to grow on laureate . In addition, Pmp47 may be involved in peroxisome proliferation, because the pmp47Delta strain contained a reduced number of peroxisomes, as judged from the fluorescence analysis of cells expressing green fluorescent protein tagged with the peroxisome targeting signal 1 (GFP-AKL).

J Biol Chem, 2000 Feb 4, 275(5), 3128 - 36
Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo function . Cofactor binding and substrate interactions; McBride AE et al.; Many eukaryotic RNA-binding proteins are modified by methylation of arginine residues . The yeast Saccharomyces cerevisiae contains one major arginine methyltransferase, Hmt1p/Rmt1p, which is not essential for normal cell growth . However, cells missing HMT1 and also bearing mutations in the mRNA-binding proteins Npl3p or Cbp80p can no longer survive, providing genetic backgrounds in which to study Hmt1p function . We now demonstrate that the catalytically active form of Hmt1p is required for its activity in vivo . Amino acid changes in the putative Hmt1p S-adenosyl-L-methionine-binding site were generated and shown to be unable to catalyze methylation of Npl3p in vitro and in vivo or to restore growth to strains that require HMT1 . In addition these mutations affect nucleocytoplasmic transport of Npl3p . A cold-sensitive mutant of Hmt1p was generated and showed reduced methylation of Npl3p, but not of other substrates, at 14 degrees C . These results define new aspects of Hmt1 and reveal the importance of its activity in vivo.

J Cell Sci, 2000 Feb, 113 ( Pt 4), 571 - 85
Polarization of cell growth in yeast; Pruyne D et al.; The actin cytoskeleton provides the structural basis for cell polarity in Saccharomyces cerevisiae as well as most other eukaryotes . In Part I of this two-part commentary, presented in the previous issue of Journal of Cell Science, we discussed the basis by which yeast establishes and maintains different states of polarity through &Rgr; GTPases and cyclin-dependent protein kinase signaling . Here we discuss how, in response to those signals, the actin cytoskeleton guides growth of the yeast cell . A polarized array of actin cables at the cell cortex is the primary structural determinant of polarity . Motors such as class V myosins use this array to transport secretory vesicles, mRNA and organelles towards growth sites, where they are anchored by a cap of cytoskeletal and regulatory proteins . Cortical actin patches enhance and maintain this polarity, probably through endocytic recycling, which allows reuse of materials and prevents continued growth at old sites . The dynamic arrangement of targeting and recycling provides flexibility for the precise control of morphogenesis.

Biochem Biophys Res Commun, 2000 Feb 5, 268(1), 112 - 7
The yeast retrotransposons Ty1 and Ty3 require the RNA Lariat debranching enzyme, Dbr1p, for efficient accumulation of reverse transcripts; Karst SM et al.; A mutant screen has been initiated to identify host genes important for the replication of retrotransposons in Saccharomyces cerevisiae . Two mutants were identified that undergo Ty1 and Ty3 transposition at <10% of the wild-type frequency . Both these mutants have deficiencies in the accumulation of full-length Ty1 and Ty3 cDNAs, although Ty proteins (including reverse transcriptase) accumulate at wild-type levels . The DBR1 gene, encoding the yeast debranching enzyme, complements both mutants . This suggests that Dbr1p is important for either reverse transcription or the stability of Ty cDNA, roles that have not been previously reported for this protein . The deficiency in accumulation of Ty cDNAs in dbr1 mutants is apparent when engineered Ty elements are expressed for short time periods (6-10 h) but is not apparent following long expression periods (>24 h) .

Mol Microbiol, 2000 Jan, 35(2), 378 - 85
Cross-talk between ammonium transporters in yeast and interference by the soybean SAT1 protein; Marini AM et al.; Ammonium uptake in the yeast Saccharomyces cerevisiae involves three membrane transporters (Mep1, -2 and -3) belonging to an evolutionarily conserved protein family that also includes the rhesus (Rh) blood group polypeptides of erythrocytes . We show here that, in the 26972c mutant defective in NH4+ transport, the Mep1 protein carrying an amino acid substitution in its cytoplasmic C-terminus trans-inhibits the closely related Mep3 protein . The same mutation introduced into Mep3 leads to loss of transport activity and this inactive form also trans-inhibits native Mep3 . Inhibition of Mep3 is post-translational and can be overcome by overexpression . These results are consistent with a direct interaction between Mep proteins, as is the case for the Rh polypeptides . The soybean GmSAT1 gene, recently cloned for its ability to complement the NH4+ transport defect of strain 26972c, has been described as an NH4+ channel protein involved in the transfer of fixed nitrogen from the bacteroid to the host plant . We show here that GmSAT1 contains a sequence homologous to the DNA-binding domain of basic helix-loop-helix (bHLH) transcription factors . We also show that GmSAT1 restores NH4+ uptake in the yeast mutant by interfering with the inhibition of Mep3 . Our results are not consistent with a direct role of GmSAT1 in ammonium transport.

Eur J Biochem, 2000 Feb, 267(3), 861 - 8
The structural basis of substrate activation in yeast pyruvate decarboxylase . A crystallographic and kinetic study; Lu G et al.; The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution . The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry . Structure analysis revealed conformational nonequivalence of the active sites . One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered . In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution . In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor . A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221 . This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer . No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule . Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme . The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur . Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.

Biochemistry (Mosc), 1999 Dec, 64(12), 1401 - 7
The {URE3} yeast prion: from genetics to biochemistry; Komar AA et al.; {URE3} is a non-Mendelian genetic element of the yeast Saccharomyces cerevisiae, an altered prion form of Ure2 protein . We show that recombinant Ure2p is a soluble protein that can assemble in vitro into dimers, tetramers, and octamers or form insoluble fibrils observed for PrP in its filamentous form or for Sup35p upon self-assembling, suggesting a similar mechanism for all prions . Computational, genetic, biochemical, and structural data allow us to specify a new boundary between the so-called prion-forming and nitrogen regulator (catalytic) domains of the protein and to map this boundary to Met-94 . We bring strong evidence that the COOH-terminal (94-354) part of the protein forms a tightly folded domain, while the NH2-terminal (1-94) part is unstructured . These domains (or various parts of these domains) were shown (by means of the two-hybrid system approach and affinity binding experiments) to interact with each other (both in vivo and in vitro) . We bring also evidence that the COOH-terminal (94-354) catalytically active part of the protein can be synthesized (both in vitro and in vivo) via an internal ribosome-binding mechanism, independently of the production of the full-length protein . We finally show that Ure2p aggregation in vivo (monitored by fluorescence of Ure2p--GFP fusion) does not necessarily give rise to {URE3} phenotype . The significance of these findings for the appearance and propagation of the yeast prion {URE3} is discussed.

Biochemistry (Mosc), 1999 Dec, 64(12), 1360 - 6
Translation termination and its regulation in eukaryotes: recent insights provided by studies in yeast; Mugnier P et al.; In protein synthesis, the arrival of one or other of the three stop codons in the ribosomal A-site triggers the binding of a release factor (RF) to the ribosome and subsequent polypeptide chain release . In eukaryotes, the RF is composed of two proteins, eRF1 and eRF3 . eRF1 is responsible for the hydrolysis of the peptidyl-tRNA, while eRF3 provides a GTP-dependent function, although its precise role remains to be defined . Recent findings on translation termination and its regulation from studies in the yeast Saccharomyces cerevisiae are reviewed and the potential role of eRF3 is discussed.

Biochemistry (Mosc), 1999 Dec, 64(12), 1337 - 41
Translation termination and yeast prions; Kisselev LL; Protein biosynthesis is the final step in the transfer of genetic information in the cell . In turn, its last step is the release of a nascent polypeptide from the ribosome . Therefore, termination of translation may be considered (if we do not take into account protein post-translational modification and folding) as a final step of the transition from genotype to phenotype through the classic DNA--RNA--protein pathway . In a narrow sense, termination of translation is the hydrolytic cleavage of peptidyl-tRNA into free tRNA and completed polypeptide chain carrying all the information encoded in the corresponding mRNA and DNA . Then the completed protein molecule is released from the ribosome and the ribosome dissociates into its components (subunits, factors, mRNA, tRNA, etc . ) . After the synthesis is completed, the polypeptide chain is folded either cotranslationally or by an additional specialized mechanism, depending on the nature of the protein, organism, and other factors . This issue of Biochemistry (Moscow) highlights from various points of view the problem of translation termination, excluding protein folding . Yeast termination factors with prion-like properties are also considered.

Mutat Res, 2000 Jan 24, 464(2), 297 - 308
Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances; Afanassiev V et al.; Yeast strains transformed with high copy number plasmids carrying the gene encoding a green fluorescent protein optimised for yeast (yEGFP3) under the control of the RAD54 or RNR2 promoter were used to investigate the activity of potentially DNA-damaging substances . The assays were performed on 96-well microtitre plates in the presence of different concentrations of the test substances . The synthesis of GFP protein was measured through the fluorescence signal and cell growth was monitored by absorption . Here, we demonstrate that this system can be used as a biosensor to assess the genotoxic potential of drugs and other chemical substances . The use of microtitre plates will enable full automation of the system and allows the inclusion of internal reference standards in each assay.

Mol Cell Biol, 2000 Feb, 20(4), 1407 - 18
A novel TATA-binding protein-binding protein, ABT1, activates basal transcription and has a yeast homolog that is essential for growth; Oda T et al.; Identification of a novel mouse nuclear protein termed activator of basal transcription 1 (mABT1) that associates with the TATA-binding protein (TBP) and enhances basal transcription activity of class II promoters is described . We also identify mABT1 homologous counterparts in Caenorhabditis elegans and Saccharomyces cerevisiae and show the homologous yeast gene to be essential for growth . The mABT1 associated with TBP in HeLa nuclear extracts and with purified mouse TBP in vitro . In addition, ectopically expressed mABT1 was coimmunoprecipitated with endogenous TBP in transfected cells . More importantly, mABT1 significantly enhanced transcription from an adenovirus major late promoter in a reconstituted cell-free system . We furthermore demonstrate that mABT1 consistently enhanced transcription from a reporter gene with a minimal core promoter as well as from reporter genes with various enhancer elements in a cotransfection assay . Taken together, these results suggest that mABT1 is a novel TBP-binding protein which can function as a basal transcription activator.

Mol Cell Biol, 2000 Feb, 20(4), 1370 - 81
Spb1p is a yeast nucleolar protein associated with Nop1p and Nop58p that is able to bind S-adenosyl-L-methionine in vitro; Pintard L et al.; We present here the characterization of SPB1, an essential yeast gene that is required for ribosome synthesis . A cold-sensitive allele for that gene (referred to here as spb1-1) had been previously isolated as a suppressor of a mutation affecting the poly(A)-binding protein gene (PAB1) and a thermosensitive allele (referred to here as spb1-2) was isolated in a search for essential genes required for gene silencing in Saccharomyces cerevisiae . The two mutants are able to suppress the deletion of PAB1, and they both present a strong reduction in their 60S ribosomal subunit content . In an spb1-2 strain grown at the restrictive temperature, processing of the 27S pre-rRNA into mature 25S rRNA and 5.8S is completely abolished and production of mature 18S is reduced, while the abnormal 23S species is accumulated . Spb1p is a 96.5-kDa protein that is localized to the nucleolus . Coimmunoprecipitation experiments show that Spb1p is associated in vivo with the nucleolar proteins Nop1p and Nop5/58p . Protein sequence analysis reveals that Spb1p possesses a putative S-adenosyl-L-methionine (AdoMet)-binding domain, which is common to the AdoMet-dependent methyltransferases . We show here that Spb1p is able to bind {(3)H}AdoMet in vitro, suggesting that it is a novel methylase, whose possible substrates will be discussed.

Mol Cell Biol, 2000 Feb, 20(4), 1361 - 9
Yeast meiosis-specific protein Hop1 binds to G4 DNA and promotes its formation; Muniyappa K et al.; DNA molecules containing stretches of contiguous guanine residues can assume a stable configuration in which planar quartets of guanine residues joined by Hoogsteen pairing appear in a stacked array . This conformation, called G4 DNA, has been implicated in several aspects of chromosome behavior including immunoglobulin gene rearrangements, promoter activation, and telomere maintenance . Moreover, the ability of the yeast SEP1 gene product to cleave DNA in a G4-DNA-dependent fashion, as well as that of the SGS1 gene product to unwind G4 DNA, has suggested a crucial role for this structure in meiotic synapsis and recombination . Here, we demonstrate that the HOP1 gene product, which plays a crucial role in the formation of synaptonemal complex in Saccharomyces cerevisiae, binds robustly to G4 DNA . The apparent dissociation constant for interaction with G4 DNA is 2 x 10(-10), indicative of binding that is about 1,000-fold stronger than to normal duplex DNA . Oligonucleotides of appropriate sequence bound Hop1 protein maximally if the DNA was first subjected to conditions favoring the formation of G4 DNA . Furthermore, incubation of unfolded oligonucleotides with Hop1 led to their transformation into G4 DNA . Methylation interference experiments confirmed that modifications blocking G4 DNA formation inhibit Hop1 binding . In contrast, neither bacterial RecA proteins that preferentially interact with GT-rich DNA nor histone H1 bound strongly to G4 DNA or induced its formation . These findings implicate specific interactions of Hop1 protein with G4 DNA in the pathway to chromosomal synapsis and recombination in meiosis.

Mol Cell Biol, 2000 Feb, 20(4), 1234 - 42
Novel WD-repeat protein Mip1p facilitates function of the meiotic regulator Mei2p in fission yeast; Shinozaki-Yabana S et al.; In fission yeast, the onset of meiosis is triggered by activation of the RNA-binding protein Mei2p . We screened for a high-copy-number suppressor of the ectopic meiosis induced by expression of an active form of Mei2p . Consequently we isolated a truncated form of a novel gene, named mip1, from a fission yeast genomic library . The mip1 gene encoded a protein of 1,313 amino acids which carried a WD-repeat motif in the C-terminal region and was apparently conserved among eukaryotes . Mip1p was cytoplasmic, and two-hybrid and immunoprecipitation analyses demonstrated that Mip1p was bound to Mei2p in vivo . Genetic evidence indicated that wild-type Mip1p was required for the function of Mei2p to induce meiosis and that the truncated form of it (Mip1-15p) dominantly interfered with Mei2p . Mip1p appeared to be involved also in conjugation, associating with Ste11p, which is a key transcription factor for sexual development . Furthermore, Mip1p was essential for cell growth, to which neither Mei2p nor Ste11p is relevant . These results suggest that Mip1p assists functional expression of a number of proteins required for proliferation and sexual development in fission yeast.

Mol Cell Biol, 2000 Feb, 20(4), 1187 - 93
Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane; Koehler CM et al.; Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex . The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p . We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p . This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form . When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p . Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis . Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p . It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex . We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

J Cell Biol, 2000 Jan 24, 148(2), 363 - 73
Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization; Lechler T et al.; The generation of cortical actin filaments is necessary for processes such as cell motility and cell polarization . Several recent studies have demonstrated that Wiskott-Aldrich syndrome protein (WASP) family proteins and the actin-related protein (Arp) 2/3 complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms . To identify other factors involved in this process, we have isolated proteins that bind to Bee1p/Las17p, the yeast WASP-like protein, by affinity chromatography and mass spectroscopic analysis . The yeast type I myosins, Myo3p and Myo5p, have both been identified as Bee1p-interacting proteins . Like Bee1p, these myosins are essential for cortical actin assembly as assayed by in vitro reconstitution of actin nucleation sites in permeabilized yeast cells . Analysis using this assay further demonstrated that the motor activity of these myosins is required for the polymerization step, and that actin polymerization depends on phosphorylation of myosin motor domain by p21-activated kinases (PAKs), downstream effectors of the small guanosine triphosphatase, Cdc42p . The type I myosins also interact with the Arp2/3 complex through a sequence at the end of the tail domain homologous to the Arp2/3-activating region of WASP-like proteins . Combined deletions of the Arp2/3-interacting domains of Bee1p and the type I myosins abolish actin nucleation sites at the cortex, suggesting that these proteins function redundantly in the activation of the Arp2/3 complex.

Yeast, 2000 Feb, 16(3), 219 - 29
Aminopeptidase yscCo-II: a new cobalt-dependent aminopeptidase from yeast-purification and biochemical characterization; Herrera-Camacho I et al.; Saccharomyces cerevisiae aminopeptidase yscCo-II (APCo-II) was purified to apparent homogeneity by gel filtration, affinity chromatography and anion-exchange chromatography . APCo-II is an hexameric cobalt-dependent metallo-enzyme with an estimated native molecular mass of 290 kDa . Enzyme activity is only detected in the presence of cobalt ions at pH 7.0 . Substrate specificity studies indicate that aminopeptidase yscCo-II cleaves only basic N-terminal residues . PMSF, Cu(2+), 1,10-phenanthroline and bestatin were found to be very strong inhibitors of aminopeptidase yscCo-II activity . Kinetic studies indicated that the enzyme has a similar K(m) and Ka(Co )(activation constant of cobalt) and, following extraction of cobalt from the enzyme, activity was recovered only after cobalt addition .

Nucleic Acids Res, 2000 Feb 15, 28(4), 1000 - 10
Statistical analysis of yeast genomic downstream sequences reveals putative polyadenylation signals; van Helden J et al.; The study of a few genes has permitted the identification of three elements that constitute a yeast polyadenyl-ation signal: the efficiency element (EE), the positioning element and the actual site for cleavage and poly-adenyl-ation . In this paper we perform an analysis of oligonucleotide composition on the sequences located downstream of the stop codon of all yeast genes . Several oligonucleotide families appear over-represented with a high significance (referred to herein as 'words') . The family with the highest over-representation includes the oligonucleotides shown experimentally to play a role as EEs . The word with the highest score is TATATA, followed, among others, by a series of single-nucleotide variants (TATGTA, TACATA, TAAATA.) and one-letter shifts (ATATAT) . A position analysis reveals that those words have a high preference to be in 3' flanks of yeast genes and there they have a very uneven distribution, with a marked peak around 35 bp after the stop codon . Of the predicted ORFs, 85% show one or more of those sequences . Similar results were obtained using a data set of EST sequences . Other clusters of over-represented words are also detected, namely T- and A-rich signals . Using these results and previously known data we propose a general model for the 3' trailers of yeast mRNAs.

Med Mycol, 1999 Dec, 37(6), 445 - 50
Analysis of the enzymatic activity of mycelial and yeast phases of Penicillium marneffei; Youngchim S et al.; The cell-associated and extracellular enzymatic activities were examined in a total of 10 Penicillium marneffei isolates . Both mycelia and yeast expressed alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities, whereas a variety of other enzyme activities, including trypsin, chymotrypsin and alpha-fucosidase were absent . There was some inter-isolate variation in both mycelia and yeast in the activities of other enzymes such as esterases and galactosidases . Enzyme activities did not change significantly over the course of culturing in three representative isolates.

J Biol Chem, 2000 Jan 28, 275(4), 2288 - 94
Yeast RNA polymerase II subunit RPB9 . Mapping of domains required for transcription elongation; Hemming SA et al.; The RPB9 subunit of RNA polymerase II regulates transcription elongation activity and is required for the action of the transcription elongation factor, TFIIS . RPB9 comprises two zinc ribbon domains joined by a conserved linker region . The C-terminal zinc ribbon is similar in sequence to that found in TFIIS . To elucidate the relationship between the structure and transcription elongation function of RPB9, we initiated a mutagenesis study on the Saccharomyces cerevisiae homologue . The individual zinc ribbon domains, in isolation or in combination, could not stimulate transcription by a polymerase lacking RPB9, pol IIDelta9 . Mutations in the N-terminal zinc ribbon had little effect on transcription activity . By contrast, mutations in the acidic loop that connects the second and third beta-strands of the C-terminal zinc ribbon were completely inactive for transcription . Interestingly, the analogous residues in TFIIS are also critical for elongation activity . A conserved charged stretch in the linker region (residues 89-95, DPTLPR) mediated the interaction with RNA polymerase II.

Science, 2000 Jan 21, 287(5452), 501 - 4
Rad6-dependent ubiquitination of histone H2B in yeast; Robzyk K et al.; Although ubiquitinated histones are present in substantial levels in vertebrate cells, the roles they play in specific biological processes and the cellular factors that regulate this modification are not well characterized . Ubiquitinated H2B (uH2B) has been identified in the yeast Saccharomyces cerevisiae, and mutation of the conserved ubiquitination site is shown to confer defects in mitotic cell growth and meiosis . uH2B was not detected in rad6 mutants, which are defective for the ubiquitin-conjugating enzyme Ubc2, thus identifying Rad6 as the major cellular activity that ubiquitinates H2B in yeast.

Yeast, 2000 Jan 30, 16(2), 177 - 87
A web site for the computational analysis of yeast regulatory sequences; van Helden J et al.; A series of computer programs were developed for the analysis of regulatory sequences, with a special focus on yeast . These tools are publicly available on the web . Basically, three classical problems can be addressed: (a) search for known regulatory patterns in the upstream regions of known genes; (b) discovery of unknown regulatory patterns within a set of upstream regions known to be co-regulated; (c) search for unknown genes potentially regulated by a known transcription factor . Each of these tasks can be performed on basis of a simple (string) or more refined (matrix) description of the regulatory patterns . A feature-map program automatically generates visual representations of the positions at which patterns were found . The site also provides a series of general utilities, such as generation of random sequence, automatic drawing of XY graphs, interconversions between sequence formats, etc . Several tools are linked together to allow their sequential utilization (piping), but each one can also be used independently by filling the web form with external data . This widens the scope of the site to the analysis of non-regulatory and/or non-yeast sequences .

Yeast, 2000 Jan 30, 16(2), 111 - 9
Calorimetric characterization of critical targets for killing and acquired thermotolerance in yeast; Obuchi K et al.; We characterized thermal behaviours of cellular components by differential scanning calorimetry (DSC) in order to investigate how Saccharomyces cerevisiae cells acquire thermotolerance after heat shock or in stationary phase . Whole-cell DSC profiles consisted of at least five endothermic components over the range 45-75 degrees C for exponentially growing, heat-shocked and stationary-phase cells . In these profiles, we attempted to localize the endothermic profiles due to denaturation of the two critical targets which were predicted by using the Arrhenius parameters of hyperthermic killing of the cells (Obuchi et al., 1998) . This prediction indicated that (a) the heat shock stabilized one family of targets and destabilized the other, while (b) arrest in stationary phase stabilized both targets . Therefore, the heat-shock response does not stabilize all cellular components, and arrest in stationary phase appears to stabilize cellular components in a different manner from the heat-shock response . It was not possible unambiguously to resolve the profiles of the critical targets in the DSC scans of whole cells . Components I (T(m)=49.7 degrees C) and II (T(m)=56.1 degrees C) may both include denaturations of critical targets 1 (T(m)=55.4 degrees C) and 2 (T(m)=53.0 degrees C) in exponential cells . Components I and II were both stabilized (T(m)=53.5 and 57.2 degrees C, respectively) in heat-shocked cells . Sub-cellular fractions suspended in 1.2 M trehalose solution, which mimics the cytosol in tolerant cells, were more stable than those in 0.6 M KCl, which mimics the cytosol in sensitive cells . The microsomal fractions in KCl and trehalose had endothermic profiles in similar temperature ranges to those predicted for sensitive and tolerant cells, respectively . This agreement suggests that the microsomal fraction may contain critical targets, and that trehalose accumulation in the heat-shocked and in the stationary phase yeast cells is a stabilizer of cellular components .

J Anim Sci, 1999 Dec, 77(12), 3371 - 6
The influence of dietary selenium as selenium yeast or sodium selenite on the concentration of selenium in the milk of Suckler cows and on the selenium status of their calves; Pehrson B et al.; The aim of this trial was to determine whether the selenium status of suckling calves could be improved by supplementing their dams' diet with organic Se instead of sodium selenite . A herd of 103 Hereford cows, which were on grass paddocks all year round, was divided into two groups . Both groups had free access to a mineral supplement that contained 30 mg of Se/kg; for one group the source of the Se was a Se yeast product, and for the other group the source was sodium selenite . The basal feed contained .02 mg of Se/kg DM . During the trial, the mean daily consumption of the mineral supplement was approximately 110 g/cow . The calving season started in the middle of March and ended in the middle of May . Blood samples were taken from 11 cows and their calves in the yeast group and from nine in the selenite group at the end of April and again at the beginning of June, and milk samples were taken at the same times . At both samplings, the concentration of Se in whole blood and the activity of glutathione peroxidase (GSH-Px) in the erythrocytes of the cows and calves in the yeast group were higher than in the samples from the animals in the selenite group . The same pattern was seen for plasma, except for the cows at the first sampling . The mean concentrations of Se in whole blood from calves in the yeast and selenite groups were 130 and 84 microg/L, respectively, and plasma concentrations were 48 and 34 microg/ L, respectively . Mean Se concentration in the milk from the yeast group (17.3 microg/L) was higher than that in milk from the selenite group (12.7 microg/L) . There were significant correlations (r = .59 to .68) between the concentrations of Se in the cow's milk or cow's whole blood compared with Se concentrations in the calves whole blood and plasma or with the erythrocyte GSH-Px activity of the calves . The Se status of the calves in the selenite group was considered to be marginal, but the status of the calves in the yeast group was considered to be adequate . Supplementation of the suckler cows' diet with organic Se in the form of Se yeast rather than sodium selenite improved the Se status of their calves when the Se was mixed into a mineral supplement containing 30 mg of Se/kg . In practice, such supplementation would probably eliminate the risk of nutritional muscular degeneration in suckling calves.

J Anim Sci, 1999 Dec, 77(12), 3365 - 70
Effect of selenate as a feed supplement to dairy cows in comparison to selenite and selenium yeast; Ortman K et al.; The main aim of this trial was to define the possible differences between selenite and selenate in their ability to increase the selenium (Se) concentration of milk, in comparison with organic Se . Dairy cows (n = 42) were fed a basal diet containing .10 to .12 mg Se/kg DM for 5 mo and were then divided into four groups of 10 or 11, as similar as possible in age and stage of lactation . During the next 84 d, the cows in three of the groups were supplemented with 3 mg of Se daily, whereas the cows in one control group remained unsupplemented . The Se supplement was given as sodium selenite, sodium selenate, or a Se yeast product . The total Se concentration of the diets varied with the cows' stage of lactation and was for the supplemented groups .24 to .31 mg/kg DM, but remained between .10 and .12 mg/kg in the control group . At the end of the trial, the mean whole blood Se concentrations in the selenite, selenate, yeast, and control groups were 138, 141, 165, and 104 microg/L, respectively . The Se concentration in plasma apparently reached a plateau level within 4 wk, at approximately 75 microg/L in the selenite group, 80 microg/L in the selenate group, and 90 microg/L in the yeast group . In the control group the mean concentration in plasma remained at approximately 50 microg/L . The increase of the activity of glutathione peroxidase (GSH-Px) in the erythrocytes was significantly higher in the supplemented groups than in the control group . The mean concentrations of Se in milk in the selenite, selenate, and yeast groups were 16.4, 16.4, and 31.2 microg/L, respectively, whereas the concentration remained at approximately 14 microg/L in the control group . The milk Se concentration reached a plateau within 1 wk after the start of Se supplementation . Dietary supplementation with selenite and selenate, thus, had only a limited effect on the Se concentration in milk, and there was no significant difference between the two inorganic compounds in any variable measured . Organic Se was much more effective than inorganic Se in increasing the concentration of Se in milk.

Genes Dev, 2000 Jan 1, 14(1), 97 - 107
ATP can be dispensable for prespliceosome formation in yeast; Perriman R et al.; The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome . We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2 . These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that they are a functional intermediate in the spliceosome assembly pathway . ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA . Loss of CUS2 does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation . Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5 . We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure.

Genes Dev, 2000 Jan 1, 14(1), 81 - 96
The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci; Frei C et al.; We have examined the cellular function of Sgs1p, a nonessential yeast DNA helicase, homologs of which are implicated in two highly debilitating hereditary human diseases (Werner's and Bloom's syndromes) . We show that Sgs1p is an integral component of the S-phase checkpoint response in yeast, which arrests cells due to DNA damage or blocked fork progression during DNA replication . DNA polepsilon and Sgs1p are found in the same epistasis group and act upstream of Rad53p to signal cell cycle arrest when DNA replication is perturbed . Sgs1p is tightly regulated through the cell cycle, accumulates in S phase and colocalizes with Rad53p in S-phase-specific foci, even in the absence of fork arrest . The association of Rad53p with a chromatin subfraction is Sgs1p dependent, suggesting an important role for the helicase in the signal-transducing pathway that monitors replication fork progression.

J Cell Sci, 2000 Feb, 113 ( Pt 3), 365 - 75
Polarization of cell growth in yeast . I . Establishment and maintenance of polarity states; Pruyne D et al.; The ability to polarize is a fundamental property of cells . The yeast Saccharomyces cerevisiae has proven to be a fertile ground for dissecting the molecular mechanisms that regulate cell polarity during growth . Here we discuss the signaling pathways that regulate polarity . In the second installment of this two-part commentary, which appears in the next issue of Journal of Cell Science, we discuss how the actin cytoskeleton responds to these signals and guides the polarity of essentially all events in the yeast cell cycle . During the cell cycle, yeast cells assume alternative states of polarized growth, which range from tightly focused apical growth to non-focused isotropic growth . RhoGTPases, and in particular Cdc42p, are essential to guiding this polarity . The distribution of Cdc42p at the cell cortex establishes cell polarity . Cyclin-dependent protein kinase, Ras, and heterotrimeric G proteins all modulate yeast cell polarity in part by altering the distribution of Cdc42p . In turn, Cdc42p generates feedback signals to these molecules in order to establish stable polarity states and coordinate cytoskeletal organization with the cell cycle . Given that many of these signaling pathways are present in both fungi and animals, they are probably ancient and conserved mechanisms for regulating polarity.

J Agric Food Chem, 2000 Jan, 48(1), 116 - 22
Release of lipids during yeast autolysis in a model wine system; Pueyo E et al.; The release of lipids during the aging of sparkling wines in contact with yeast can influence wine sensory attributes and, especially, foam characteristics . Model systems allow study of the autolysis process in a reasonable period of time compared to natural conditions, at which it can last several months . In this paper, the release of the different classes of lipids during the autolysis of three commercial yeast strains in a model wine medium has been monitored . Due to the absence of accurate quantitative methods, an HPLC method for separating and quantifying the different neutral and polar yeast lipid classes was developed . Lipids were eluted through a YMC PVA-Sil column with a complex solvent mixture . Detection was carried out with a light-scattering detector . The yeasts were suspended in the model wine buffer and incubated at 30 degrees C for up to 12 days . A release of triacylglycerols, 1,3-diacylglycerols, 2-monoacylglycerols, free fatty acids, sterol esters, and sterols was observed over the first 2 days, a period that corresponded to the maximum loss of yeast viability . A decrease in most of these lipids was observed from day 2, possibly indicative of the release of yeast hydrolytic enzymes due to the breakdown of the cell wall . Phospholipids were not detected in any of the autolysates . The mean lipid content in the autolysates as a percentage of the total lipid content in the yeasts was 8.6% for sterol esters, 3.8% for sterols, 2% for triacylglycerols, and <2% for 1,3-diacylglycerols and free fatty acids.

Neurobiol Aging, 1999 Sep-Oct, 20(5), 479 - 86
Mutations in signal transduction proteins increase stress resistance and longevity in yeast, nematodes, fruit flies, and mammalian neuronal cells; Longo VD; Mutations in Ras and other signal transduction proteins increase survival and resistance to oxidative stress and starvation in stationary phase yeast, nematodes, fruit flies, and in neuronal PC12 cells . The chronological life span of yeast, based on the survival of nondividing cells in stationary phase, has allowed the identification and characterization of long-lived strains with mutations in the G-protein Ras2 . This paradigm was also used to identify the in vivo sources and targets of reactive oxygen species and to examine the role of antioxidant enzymes in the longevity of yeast . I will review this model system and discuss the striking phenotypic similarities between long-lived mutants ranging from yeast to mammalian neuronal cells . Taken together, the published studies suggest that survival may be regulated by similar fundamental mechanisms in many eukaryotes and that simple model systems will contribute to our understanding of the aging process in mammals.

Nucleic Acids Res, 2000 Feb 1, 28(3), 835 - 42
Transcription-coupled DNA repair in yeast transcription factor IIE (TFIIE) mutants; Lommel L et al.; We examined the role of yeast transcription initiation factor IIE (TFIIE) in eukaryotic transcription-coupled repair (TCR), the preferential removal of DNA damage from the transcribed strands of genes over non-transcribed sequences . TFIIE can recruit the transcription initiation/repair factor TFIIH to the RNA polymerase II (RNA pol II) initiation complex to facilitate promoter clearance . Following exposure to UV radiation, the RNA pol II elongation complex is blocked at sites of UV-induced DNA damage, and may be recognized by nucleotide excision repair proteins, thus enabling TCR . The TFA1 gene encodes the large subunit of TFIIE . We determined how DNA repair is affected by TFA1 conditional mutations . In particular, we find proficient TCR in a heat-sensitive tfa1 mutant at the non-permissive temperature during which growth is inhibited and overall RNA pol II transcription is reported to be inhibited . We demonstrate that transcription of the RPB2 gene was reduced, but readily detectable, in the heat-sensitive tfa1 mutant at the non-permissive temperature and thereby prove that TCR does occur in an expressed gene in the absence of TFIIE in vivo . We demonstrate that TCR occurs even at low levels of transcription.

Nucleic Acids Res, 2000 Feb 1, 28(3), 809 - 17
Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits; Albert TK et al.; The yeast CCR4-NOT protein complex is a global regulator of RNA polymerase II transcription . It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional proteins like yeast CAF1 . Here we report the isolation of cDNAs encoding human NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF . Analysis of their mRNA levels in different human tissues reveals a common ubiquitous expression pattern . A multitude of two-hybrid interactions among the human cDNAs suggest that their encoded proteins also form a complex in mammalian cells . Functional conservation of these proteins throughout evolution is supported by the observation that the isolated human NOT3 and NOT4 cDNAs can partially com-plement corresponding not mutations in yeast . Interestingly, human CALIF is highly homologous to, although clearly different from, a recently described human CAF1 protein . Conserved interactions of this factor with both NOT and CCR4 proteins and co-immunoprecipitation experiments suggest that CALIF is a bona fide component of the human CCR4-NOT complex.

Mol Biol Cell, 2000 Jan, 11(1), 369 - 91
Kinetic analysis of a molecular model of the budding yeast cell cycle; Chen KC et al.; The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism . In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1-3 and Clb1-6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation . These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation . Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities . Model in hand, we examine the molecular events controlling "Start" (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and "Finish" (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants . The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast.

Mol Biol Cell, 2000 Jan, 11(1), 305 - 23
Vps52p, Vps53p, and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi; Conibear E et al.; The late Golgi of the yeast Saccharomyces cerevisiae receives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized . We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment . Mutation of VPS52, VPS53, or VPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected . A vps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport . Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients . This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy . Together, the phenotypic and biochemical data suggest that VPS52, VPS53, and VPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment . The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.

Mol Biol Cell, 2000 Jan, 11(1), 171 - 82
Organization of the yeast Golgi complex into at least four functionally distinct compartments; Brigance WT et al.; Pro-alpha-factor (pro-alphaf) is posttranslationally modified in the yeast Golgi complex by the addition of alpha1,6-, alpha1,2-, and alpha1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event . Previous work has indicated that the alpha1,6- and alpha1,3-mannosylation and Kex2p-dependent processing of pro-alphaf are initiated in three distinct compartments of the Golgi complex . Here, we present evidence that alpha1,2-mannosylation of pro-alphaf is also initiated in a distinct Golgi compartment . Linkage-specific antisera and an endo-alpha1,6-D-mannanase (endoM) were used to quantitate the amount of each pro-alphaf intermediate during transport through the Golgi complex . We found that alpha1,6-, alpha1,2-, and alpha1,3-mannose were sequentially added to pro-alphaf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step . The Sec18p dependence implies that a transport event was required between each modification event . In addition, most of the Golgi-modified pro-alphaf that accumulated in brefeldin A-treated cells received only alpha1,6-mannosylation as did approximately 50% of pro-alphaf transported to the Golgi in vitro . This further supports the presence of an early Golgi compartment that houses an alpha1,6-mannosyltransferase but lacks alpha1,2-mannosyltransferase activity in vivo . We propose that the alpha1,6-, alpha1,2-, and alpha1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial, trans, and trans-Golgi network of the yeast Golgi complex, respectively.

Mol Biol Cell, 2000 Jan, 11(1), 23 - 38
Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes; Lewis MJ et al.; Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane . We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p . GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi . Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain . Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific . Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome-Golgi traffic . Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface . The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p . We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.

Vestn Ross Akad Med Nauk, 1999, (11), 54 - 6
{Synthetic media for cultivation of lipophilic yeast malassezia spp}; Arzumanian VG; The capacity of the lipophilic yeast Malassezia spp . (Pityrosporum spp.) to grown in a synthetic nutritional media has been studied . The modified Dixon's medium was taken as a prototype for preparing the medium containing amino acid, Tween, and an emulsifier as carbon and energy sources . The isolates of M . sympodialis have demonstrated to relatively more rapidly grow in the medium than those of M . globosa.

Mol Microbiol, 2000 Jan, 35(1), 15 - 31
Recruitment of the yeast MADS-box proteins, ArgRI and Mcm1 by the pleiotropic factor ArgRIII is required for their stability; El Bakkoury M et al.; Regulation of arginine metabolism requires the integrity of four regulatory proteins, ArgRI, ArgRII, ArgRIII and Mcm1 . To characterize further the interactions between the different proteins, we used the two-hybrid system, which showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRIII, without an arginine requirement . To define the interacting domains of ArgRI and Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA-binding domain of Gal4 (GBD) and created mutations in GBD-ArgRI and GBD-Mcm1 . The putative alpha helix present in the MADS-box domain of ArgRI and Mcm1 is their major region of interaction with ArgRIII . Interactions between the two MADS-box proteins and ArgRIII were confirmed using affinity chromatography . The requirement for ArgRIII in the control of arginine metabolism can be bypassed in vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that ArgRIII is not present in the protein complex formed with the 'arginine boxes' . We show that the impairment of arginine regulation in an argRIII deletant strain is a result of a lack of stability of ArgRI and Mcm1 . A mutation in ArgRI, impairing its interaction with ArgRIII, leads to an unstable ArgRI protein in a wild-type strain . ArgRIII integrity is crucial for Mcm1 function, as shown by the marked decreased expression of five genes controlled by Mcm1 . However, ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression of Mcm1 does not compensate some physiological defects observed in an argRIII deletant strain.

J Bacteriol, 2000 Jan, 182(2), 540 - 2
The HTR1 gene is a dominant negative mutant allele of MTH1 and blocks Snf3- and Rgt2-dependent glucose signaling in yeast; Schulte F et al.; Saccharomyces cerevisiae HTR1 mutants are severely impaired in the uptake of glucose . We have cloned dominant HTR1 mutant alleles and show that they encode mutant forms of the Mth1 protein . Mth1 is shown to be involved in carbon source-dependent regulation of its own, invertase and hexose transporter gene expression . The mutant forms block the transduction of the Snf3- and Rgt2-mediated glucose signals upstream of the Rgt1 transcriptional regulator.

Arch Microbiol, 2000 Nov, 174(5), 353 - 61
Hydroxylation of biphenyl by the yeast Trichosporon mucoides; Sietmann R et al.; Hydroxylation of biphenyl by the dibenzofuran-degrading yeast Trichosporon mucoides SBUG 801 was studied . Glucose-grown cells degraded 40% of the biphenyl added within the first 24 h of incubation . The first step in the biotransformation pathway was the monohydroxylation of the biaryl compound to produce 2-, 3-, and 4-hydroxybiphenyl . Further oxidation produced seven dihydroxylated intermediates; the second hydroxyl group was added either on the aromatic ring already hydroxylated or on the second ring . Of all metabolites, 2,5-dihydroxybiphenyl accumulated in the supernatant in the highest concentration . The initial hydroxylation favors the 4-position to produce 4-hydroxybiphenyl, which is subsequently hydroxylated to form 3,4-dihydroxybiphenyl . When biphenyl was replaced as a substrate by 4-hydroxybiphenyl, further hydroxylation of the intermediate 3,4-dihydroxybiphenyl resulted in 3,4,4'-trihydroxybiphenyl . Incubation of T . mucoides with biphenyl and 18O2 indicated a monooxygenase-catalyzed reaction in the oxidation of biphenyl . The hydroxylation was inhibited by 1-aminobenzotriazole and metyrapone, known cytochrome P450 inhibitors . These results are very similar to those observed in the biotransformation of biphenyl in mammals.

Immunol Invest, 2000 Nov, 29(4), 355 - 64
Mode of action of plasmolysed yeast on lymphocytes under microgravity stress; Joller PW et al.; A newly developed device to simulate microgravity for space biological investigations under laboratory conditions allowed us to apply a reproducible environmental stress on immunologically active cells . Cell proliferation, soluble IL-2 receptor in the culture supernatant, lymphocyte surface activation markers like CD25 (IL-2R), CD69 and HLA-Dr were the endpoints measured . Untreated donor lymphocyte reactions under microgravity were compared to the same cells treated with an immunomodulator from herbal plasmolysed yeast (Bio-Strath Food Supplement) . The main finding is the enhancement of the proliferation inhibition under microgravitational stress by the herbal plasmolysed yeast.

Nucleic Acids Res, 2000 Dec 15, 28(24), 4956 - 63
A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA; Ling F et al.; A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30 degrees C and shows extensive vegetative petite induction by UV irradiation at 30 degrees C or when cultivated at a higher temperature (37 degrees C) . It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration . Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay . mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells . Soon after a temperature shift from 30 to 37 degrees C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells . Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37 degrees C . Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level . These observations are consistent with MHR1 having a possible role in mtDNA repair.

Genetics, 2000 Jan, 154(1), 133 - 46
Regulation of mitotic homeologous recombination in yeast . Functions of mismatch repair and nucleotide excision repair genes; Nicholson A et al.; The Saccharomyces cerevisiae homologs of the bacterial mismatch repair proteins MutS and MutL correct replication errors and prevent recombination between homeologous (nonidentical) sequences . Previously, we demonstrated that Msh2p, Msh3p, and Pms1p regulate recombination between 91% identical inverted repeats, and here use the same substrates to show that Mlh1p and Msh6p have important antirecombination roles . In addition, substrates containing defined types of mismatches (base-base mismatches; 1-, 4-, or 12-nt insertion/deletion loops; or 18-nt palindromes) were used to examine recognition of these mismatches in mitotic recombination intermediates . Msh2p was required for recognition of all types of mismatches, whereas Msh6p recognized only base-base mismatches and 1-nt insertion/deletion loops . Msh3p was involved in recognition of the palindrome and all loops, but also had an unexpected antirecombination role when the potential heteroduplex contained only base-base mismatches . In contrast to their similar antimutator roles, Pms1p consistently inhibited recombination to a lesser degree than did Msh2p . In addition to the yeast MutS and MutL homologs, the exonuclease Exo1p and the nucleotide excision repair proteins Rad1p and Rad10p were found to have roles in inhibiting recombination between mismatched substrates.

Genetics, 2000 Jan, 154(1), 61 - 71
Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression; Ben-Yehuda S et al.; Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction . It was found recently that PRP17 is identical to the cell division cycle CDC40 gene . cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication . Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature . In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene . PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction . We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein . In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system . Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction . On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.

Mol Gen Genet, 1999 Dec, 262(4-5), 589 - 99
Yeast genes GIS1-4: multicopy suppressors of the Gal- phenotype of snf1 mig1 srb8/10/11 cells; Balciunas D et al.; Cyclin C and the cyclin C-dependent protein kinase are associated with the RNA polymerase II Mediator complex, which regulates initiation of transcription in response to signals from activators and repressors bound to upstream promoter elements . Disruption of the corresponding genes, SRB11 and SRB10, in budding yeast causes a reduction in expression of the GAL genes, which is particularly pronounced in a mig1 snf1 background . We have screened two yeast genomic libraries for genes that can suppress this phenotype when overexpressed . Seven suppressor genes were identified, GIS1-7 . GIS1 encodes one of two related zinc-finger proteins, which also share two other highly conserved domains present in several eukaryotic transcription factors . GIS2 encodes a homologue of the mammalian CNBP and fission yeast Byr3 proteins . GIS3 and GIS4 predict proteins with no obvious similarities to any known proteins . GIS5-7 are identical to the previously described genes PDE2, SGE1 and TUB3, respectively . None of the suppressor genes seem to be involved in Mediator function . Instead, we find that the GIS1, GIS2 and GIS4 genes interact with the CDC25 gene, indicating a possible involvement of these genes in the RAS/cAMP signaling pathway.

Blood, 2000 Jan 15, 95(2), 719 - 20
Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system; Reuter T et al.; Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown . Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins . They also obtained evidence for a weak interaction between FANCA and FANCC . Neither FANCA nor FANCC was found to interact with itself . These results support the notion of a functional association between the FA gene products . (Blood . 2000;95:719-720)

Biochim Biophys Acta, 2000 Jan 10, 1456(2-3), 67 - 76
Interactions of arsenate, sulfate and phosphate with yeast mitochondria; Cortes P et al.; In the presence of K(+), addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane potential (DeltaPsi) and this is prevented by millimolar concentrations of phosphate (PO(4)) . Different monovalent and polyvalent anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae . Only arsenate (AsO(4)) and sulfate (SO(4)) were as efficient as PO(4) to protect mitochondria against the K(+) mediated swelling, depletion of the DeltaPsi, and decrease in the ratio of uncoupled state to state 4 respiration rates . Protection by PO(4), SO(4) or AsO(4) was inhibited by mersalyl, suggesting that these anions interact with a site located in the matrix side . In addition, the effects of SO(4) and AsO(4) on the F(1)F(0)-ATPase were tested: both SO(4) and AsO(4) inhibited the synthesis of ATP following competitive kinetics against PO(4) and non-competitive kinetics against ADP . The mersalyl sensitive uptake of (32)PO(4) was not inhibited by SO(4) or AsO(4), suggesting that the synthesis of ATP was inhibited at the F(1)F(0)-ATPase . The hydrolysis of ATP was not inhibited, only a stimulation was observed when AsO(4) or sulfite (SO(3)) were added . It is suggested that the structure and charge similarities of PO(4), AsO(4) and SO(4) result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site, occupation by any of these three anions results in protection against uncoupling by K(+); at the second site, in the F(1)F(0)-ATPase, AsO(4) and SO(4) compete for binding against PO(4) leading to inhibition of the synthesis of ATP.

J Biol Chem, 2000 Jan 14, 275(2), 1275 - 8
Nuclease-hypersensitive chromatin formed by a CpG island in human DNA cloned as an artificial chromosome in yeast; Mucha M et al.; CpG islands are mostly unmethylated GC-, and CpG-rich chromosomal segments overlapping promoter sequences in all housekeeping and many tissue-specific genes in vertebrates . Typically, these islands show an open chromatin structure, low in histone H1 and rich in acetylated histones . We have previously found that the island-like CGCG-rich sites in human DNA are hypersensitive to DNase I upon cloning in Saccharomyces cerevisiae . Here we studied, with a higher resolution, the chromatin formed in yeast by one such site, the CpG island accompanying the human glucose-6-phosphate dehydrogenase gene . We have found two strong hypersensitive sites and several positioned nucleosomes flanking the island despite the absence in yeast of such chromatin fiber-shaping factors as histone H1, methyltransferase, and the tissue-specific transcription factors . This finding, together with similar observations from our laboratories and others supports the idea that variations in GC and/or CpG content substantially contribute to the DNA sequence features modulating the structure of the chromatin . The composition-dependent fluctuations in the accessibility of DNA in the chromatin may constitute an evolutionary advantage and may explain the surprising compositional selection that acts in both the coding and non-coding segments of some genes during mammalian evolution.

J Mol Biol, 2000 Jan 21, 295(3), 393 - 409
Role of an alpha-helical bulge in the yeast heat shock transcription factor; Hardy JA et al.; The heat shock transcription factor (HSF) is the master transcriptional regulator of the heat shock response . The identity of a majority of the genes controlled by HSF and the circumstances under which HSF becomes induced are known, but the details of the mechanism by which HSF is able to sense and respond to heat remains an enigma . For example, it is unclear whether HSF senses the heat shock directly or requires ancillary interactions from a heat-induced signaling pathway . We present the analysis of a series of mutations in an alpha-helical bulge in the DNA-binding domain of HSF . Deletion of residues in this bulged region increases the overall activity of the protein . Yeast containing the deletion mutant HSF are able to survive growth temperatures that are lethal to yeast containing wild-type HSF, and they are also constitutively thermotolerant . The increase in activity can be measured as an increase in both constitutive and induced transcriptional activity . The mutant proteins bind DNA more tightly than the wild-type protein does, but this is unlikely to account fully for the increase in transcriptional activity as yeast HSF is constitutively bound to its binding site in vivo . The stability of the mutant proteins to thermal denaturation is lower than wild-type, though their native-state structures are still well-folded . Therefore, the mutants may be structurally analogous to the heat-induced state of HSF, and suggest that the DNA-binding domain of HSF may be capable of sensing heat shock directly .

J Immunol, 2000 Jan 15, 164(2), 812 - 24
High-level rearrangement and transcription of yeast artificial chromosome-based mouse Ig kappa transgenes containing distal regions of the contig; Li S et al.; The mouse Ig kappa L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved . Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon V kappa-J kappa joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci . The largest minilocus (290 kb) possessed all members of the upstream V kappa 2 gene family including their entire 5' and 3' flanking sequences, along with one member of a downstream V kappa 21 gene family . In addition, again using yeast artificial chromosome-based technology, we created Ig kappa miniloci that contained differing lengths of sequences 5' of the most distal V kappa 2 gene family member . In transgenic mice, Ig kappa miniloci exhibited position-independent and copy number-dependent germline transcription . Ig kappa miniloci were rearranged in tissue and developmental stage-specific manners . The levels of rearrangement and transcription of the distal and proximal V kappa gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects . The minilocus that contained the longest 5' region exhibited significantly greater recombination of the upstream V kappa 2 genes but not the downstream V kappa 21 gene, providing evidence for a local recombination stimulating element . These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Ig kappa gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.

FEBS Lett, 1999 Dec 3, 462(3), 411 - 5
Properties of the human long and short isoforms of the uncoupling protein-3 expressed in yeast cells; Hinz W et al.; Two splice variants of the human uncoupling protein-3 (UCP3L and UCP3S) are highly expressed in skeletal muscle . The properties of UCP3L and S have been compared to those of UCP1 in a heterologous yeast expression system under the control of the galactose promoter . Both UCP3 isoforms were found to strongly impair the coupling efficiency of respiring cells thus resulting in increased thermogenesis . The uncoupling properties of both UCP3L and S could be clearly demonstrated also in isolated yeast mitochondria both in terms of coupled respiration and in the capacity to polarize the inner membrane in conditions of limited substrate availability . Contrary to what was observed with mitochondria containing UCP1, millimolar GDP and ATP had little if any effect on the uncoupling activity of UCP3 . A very marked uncoupling of whole cells and isolated mitochondria was observed at very low expression levels of UCP3S indicating that the short isoform is more active than the long one.

FEBS Lett, 1999 Dec 3, 462(3), 313 - 6
Metabolite transport in isolated yeast mitochondria: fumarate/malate and succinate/malate antiports; Pallotta ML et al.; In this study, we investigated the metabolite permeability of isolated coupled Saccharomyces cerevisiae mitochondria . The occurrence of a fumarate/malate antiporter activity was shown . The activity differs from that of the dicarboxylate carrier (which catalyses the succinate/malate antiport) in (a) kinetics (Km and Vmax values are about 27 microM and 22 nmol min(-1) mg protein(-1) and 70 microM and 4 nmol min(-1) mg protein(-1), respectively), (b) sensitivity to inhibitors, (c) Ki for the competitive inhibitor phenylsuccinate and (d) pH profiles.

Yeast, 2000 Jan 15, 16(1), 49 - 55
Differential fates of invertase mutants in the yeast endoplasmic reticulum; McCracken AA et al.; A number of proteins have been identified as substrates for endoplasmic reticulum (ER)-associated protein degradation (ERAD) and we describe here a new model substrate with which to study this process . Two secretion-defective forms of yeast invertase that accumulated in the ER to greatly different levels were examined: Suc2-538p levels were low, while Suc2-533p was present in high amounts . Because Suc2-533p and Suc2-538p mRNA levels were comparable, we examined whether Suc2-538p was targeted for degradation . Both mutant polypeptide levels were unaffected in a yeast strain deficient in vacuolar protease activity and, additionally, we showed that Suc2-538p was stabilized in ERAD-deficient strains, demonstrating that Suc2-538p was a substrate for ERAD .

Genes Cells, 1999 Nov, 4(11), 627 - 42
The Pho85 kinase, a member of the yeast cyclin-dependent kinase (Cdk) family, has a regulation mechanism different from Cdks functioning throughout the cell cycle; Nishizawa M et al.; BACKGROUND: The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85p) which is highly homologous to the Cdc28 kinase (Cdc28p) . Although the two kinases share a 51% identity and their functional domains are well conserved, PHO85 fails to replace CDC28 . Pho85p forms complexes with G1-cyclin homologues, including Pcl1p, Pcl2p and Pcl9p, and is thought to be involved in the cell-cycle regulation at G1 and the end of M . By analysing the genetic and biochemical properties of Pho85p, we studied whether the regulation of Pho85p activity is similar to other cyclin-dependent kinases (Cdks) directly involved in cell cycle regulation . RESULTS: A functional analysis of various Pho85 mutants revealed that E53 in the PSTAIRE sequence was important for Pho85p function . On the other hand, residues in the T-loop including S166, S167 and E168, appeared dispensable for Pho85p function, suggesting that the phosphorylation of S166, corresponding to T161 of Cdc2p and T169 of Cdc28p, was not required for the kinase activity of Pho85p . Instead, we found that phosphorylation of Y18, corresponding to Y15 of Cdc2p and Y19 of Cdc28p, may be important for the binding of Pho80p but not of Pcl1p, suggesting that tyrosine phosphorylation may function as a signal which discriminates various Pho85-cyclins . CONCLUSION: In Cdks functioning throughout the cell cycle, tyrosine phosphorylation is inhibitory to the activation of kinase, whereas the phosphorylation of threonine in the T-loop is essential for activation . Our finding indicates that the regulation mechanism of Pho85p activation appears to be distinct from these Cdks.

Genes Cells, 1999 Nov, 4(11), 619 - 25
Bloom's syndrome gene suppresses premature ageing caused by Sgs1 deficiency in yeast; Heo SJ et al.; BACKGROUND: Bloom's syndrome (BS) is an autosomal recessive disorder causing short stature, immunodeficiency, and an increased risk of cancer . Increased rates of sister chromatid exchange and chromosomal aberration have been observed in cells having defects in the BLM gene . Among five kinds of human RecQ helicases cloned, the mutations in WRN and RecQL4 have been known as the causes of premature ageing . Little is, however, known about the function of BLM helicase in ageing . RESULTS: We show that human BLM, but not WRN can prevent the premature ageing and the increased homologous recombination at the rDNA loci caused by sgs1 mutation . Unexpectedly, the levels of ERCs (extrachromosomal rDNA circles), the products of homologous recombination, formed in 7-generation cells of the wild-type or the sgs1:BLM strain were comparable with those of the sgs1 or the sgs1:WRN age-matched-old cells . CONCLUSION: These results imply that BLM helicase may have an important role in human ageing . In addition, these data suggest that the accumulated ERCs per se may be not the cause of premature ageing in yeast, inconsistent with the model proposed by Sinclair & Guarente . We discuss a new model, which explains how Sgs1 or BLM helicase suppresses premature ageing in yeast.

EMBO J, 2000 Jan 4, 19(1), 37 - 47
The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex; Ohnacker M et al.; In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)-binding protein I (Pab1p) . We report the characterization of Pfs2p, a WD-repeat protein previously identified in a multiprotein complex carrying PF I-Pap1p activity . The 3'-end-processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation . With a one-step affinity purification method that exploits protein A-tagged Pfs2p, we showed that this protein is part of a CF II-PF I complex . Pull-down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II-PF I and CF IA . These results show that Pfs2p plays an essential role in 3'-end formation by bridging different processing factors and thereby promoting the assembly of the processing complex.

Environ Mol Mutagen, 1999, 34(4), 285 - 90
Polychlorinated biphenyls induce meiotic length mutations at the human minisatellite MS32 in yeast; Appelgren H et al.; Polychlorinated biphenyls (PCBs) are lipophilic compounds, several of which are toxic and carcinogenic . Complex mixtures of PCBs (e.g., Aroclor) have been widely used in the industry . The persistence of PCBs, in combination with poor waste management, has led to a large-scale distribution of PCBs in the biosphere . The toxic and carcinogenic effects of PCBs are poorly understood, but are suggested to be associated with Ah receptor binding and induction of the Ah-gene battery . We have previously shown that a higher-chlorinated PCB mixture, Aroclor 1254, significantly increased the germline mutation rate at the mouse minisatellite PC-1 . We have recently developed an in vitro model system to study and characterize spontaneous and induced meiotic mutations in human minisatellites integrated in yeast . Here, for the first time, we have used this model system to show that chemicals, in this case Aroclor 1254, can induce meiotic length mutations at the human minisatellite MS32 in a yeast strain harboring 38- and 42-repeat-unit alleles . The results also show that the size distribution of mutant MS32 alleles differs between PCB and the control, with a larger proportion of mutant allele sizes below 29 repeat units in the PCB series . These alleles were not structurally different from the alleles of the same size in the control . We conclude that PCBs induce minisatellite mutations in meiosis and have recombinogenic properties, and that the mutations are induced in an Ah receptor-independent manner . The induction of minisatellite mutations in meiosis as an indication of genomic damage must be taken into account in the risk assessment of PCBs and other environmental contaminants .

FEBS Lett, 1999 Dec 31, 464(3), 143 - 7
Evolutionally conserved plant homologue of the Bax inhibitor-1 (BI-1) gene capable of suppressing Bax-induced cell death in yeast(1); Kawai M et al.; The plant homologue of Bax Inhibitor-1, a gene described to suppress the cell death induced by Bax gene expression in yeast, was isolated from Oryza sativa L . (rice) and Arabidopsis . The amino acid sequence of the predicted protein was well conserved in both animal and plant (45% in amino acids) and contained six or seven membrane-spanning segments . Northern blot analysis showed that OsBI-1 transcripts were present in all tissues examined . The OsBI-1 cDNA suppressed cell death induced by mammalian Bax in yeast, suggesting functional conservation of this BI-1 homologue in the plant kingdom.

Proc Natl Acad Sci U S A, 2000 Jan 4, 97(1), 121 - 6
Yeast flavin-containing monooxygenase is induced by the unfolded protein response; Suh JK et al.; Flavin-containing monooxygenase from yeast (yFMO) carries out the O(2)- and NADPH-dependent oxidation of biological thiols, including oxidizing glutathione to glutathione disulfide . FMO provides a large fraction of the oxidizing necessary for proper folding of disulfide bond-containing proteins; deletion of the enzyme reduces proper folding of endogenous carboxypeptidase Y by about 40% . The enzyme is not essential to cell viability because other enzymes can generate a significant fraction of the oxidizing equivalents required by the cell . However, yFMO is vital to the yeast response to reductive stress . FMO1 deletion mutants grow poorly under reductive stress, and carboxypeptidase Y activity is less than 10% of that in a stressed wild type . The FMO1 gene appears to be under control of an unfolded protein response element and is inducible by factors, such as reductive stress, that elicit the unfolded protein response . Reductive stress can increase yFMO activity at least 6-fold . This increased activity allows the cell to process endogenous disulfide bond-containing proteins and also to allow correct folding of disulfide-bonded proteins expressed from multicopy plasmids . The unfolded protein response is mediated by the Hac1p transcription factor that mediates virtually all of the induction of yFMO triggered by exogenous reducing agents.

J Biol Chem, 2000 Jan 7, 275(1), 521 - 9
Distinct domains within yeast Sec61p involved in post-translational translocation and protein dislocation; Wilkinson BM et al.; The translocation of secretory polypeptides into and across the membrane of the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane . Recent data also suggest that misfolded or unassembled polypeptides exit the ER via the translocon for degradation by the cytosolic ubiquitin/proteasome pathway . Sec61p is a highly conserved multispanning membrane protein that constitutes a core component of the translocon . We have found that the essential function of the Saccharomyces cerevisiae Sec61p is retained upon deletion of either of two internal regions that include transmembrane domains 2 and 3, respectively . However, a deletion mutation encompassing both of these domains was found to be nonfunctional . Characterization of yeast mutants expressing the viable deletion alleles of Sec61p has revealed defects in post-translational translocation . In addition, the transmembrane domain 3 deletion mutant is induced for the unfolded protein response and is defective in the dislocation of a misfolded ER protein . These data demonstrate that the various activities of Sec61p can be functionally dissected . In particular, the transmembrane domain 2 region plays a role in post-translational translocation that is required neither for cotranslational translocation nor for protein dislocation.

J Biol Chem, 2000 Jan 7, 275(1), 467 - 71
Purification of protein A-tagged yeast ran reveals association with a novel karyopherin beta family member, Pdr6p; Lau D et al.; The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport . GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant . After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g . Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran . Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo . Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.

J Biol Chem, 2000 Jan 7, 275(1), 255 - 60
Cysteine scanning mutagenesis of the noncatalytic nucleotide binding site of the yeast V-ATPase; Vasilyeva E et al.; To investigate residues involved in the formation of the noncatalytic nucleotide binding sites of the vacuolar proton-translocating adenosine triphosphatase (V-ATPase), cysteine scanning mutagenesis of the VMA2 gene that encodes the B subunit in yeast was performed . Replacement of the single endogenous cysteine residue at position 188 gave rise to a Cys-less form of the B subunit (Vma2p) which had near wild-type levels of activity and which was used in the construction of 16 single cysteine-containing mutants . The ability of adenine nucleotides to prevent reaction of the introduced cysteine residues with the sulfhydryl reagent 3-(N-maleimidopropionyl)biocytin (biotin-maleimide) was evaluated by Western blot . Biotin-maleimide labeling of the purified V-ATPase from the wild-type and the mutants S152C, L178C, N181C, A184C, and T279C was reduced after reaction with the nucleotide analog 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (BzATP) . These results suggest the proximity of these residues to the nucleotide binding site on the B subunit . In addition, we have examined the level of endogenous nucleotide bound to the wild-type V-ATPase and to a mutant (the A subunit mutant R483Q) which is postulated to be altered at the noncatalytic site and which displays a marked nonlinearity in ATP hydrolysis (MacLeod, K . J., Vasilyeva, E., Baleja, J . D., and Forgac, M . (1998) J . Biol . Chem . 273, 150-156) . The R483Q mutant contained 2.6 mol of ATP/mol of V-ATPase compared with the wild-type enzyme, which contained 0.8 mol of ATP/mol of V-ATPase . These results suggest that binding of additional ATP to the noncatalytic sites may modulate the catalytic activity of the enzyme.

J Biol Chem, 2000 Jan 7, 275(1), 248 - 54
The coiled coil dimerization element of the yeast transcriptional activator Hap1, a Gal4 family member, is dispensable for DNA binding but differentially affects transcriptional activation; Hach A et al.; The heme activator protein Hap1 is a member of the yeast Gal4 family, which consists of transcription factors with a conserved Zn(2)Cys(6) cluster that recognizes a CGG triplet . Many members of the Gal4 family contain a coiled coil dimerization element and bind symmetrically to DNA as homodimers . However, Hap1 possesses two unique properties . First, Hap1 binds asymmetrically to a direct repeat of two CGG triplets . Second, Hap1 binds to two classes of DNA elements, UAS1/CYC1 and UAS/CYC7, and permits differential transcriptional activation at these sites . Here we determined the residues of the Hap1 dimerization domain critical for DNA binding and differential transcriptional activation . We found that the Hap1 dimerization domain is composed of functionally redundant elements that can substitute each other in DNA binding and transcriptional activation . Remarkably, deletion of the coiled coil dimerization element did not severely diminish DNA binding and transcriptional activation at UAS1/CYC1 but completely abolished transcriptional activation at UAS/CYC7 . Furthermore, Ala substitutions in the dimerization element selectively diminished transcriptional activation at UAS/CYC7 . These results strongly suggest that the coiled coil dimerization element is responsible for differential transcriptional activation at UAS1/CYC1 and UAS/CYC7 and for making contacts with a putative coactivator or part of the transcription machinery.

Biol Chem, 1999 Nov, 380(11), 1341 - 5
The anti-estrogen hydroxytamoxifen is a potent antagonist in a novel yeast system; Liu JW et al.; The budding yeast Saccharomyces cerevisiae has been used extensively as a biological 'test tube' to study the regulation of the human estrogen receptor (ER) alpha . However, anti-estrogens, which are of great importance as therapeutic agents and research tools, fail to antagonize the activation by estrogen in yeast . Here, we have surveyed the antagonistic potential of five different anti-estrogens of diverse chemical nature . While they all act as agonists for wild-type ERalpha, we have established a novel yeast assay system for anti-estrogens, in which at least the commonly used anti-estrogen hydroxytamoxifen is a potent antagonist.

Annu Rev Cell Dev Biol, 1999, 15, 365 - 91
Cell polarity in yeast; Chant J; Subcellular asymmetry, cell polarity, is fundamental to the diverse specialized functions of eukaryotic cells . In yeast, cell polarization is essential to division and mating . As a result, this highly accessible experimental system serves as a paradigm for deciphering the molecular mechanisms underlying the generation of polarity . Beyond yeast, cell polarity is essential to the partitioning of cell fate in embryonic development, the generation of axons and their guidance during neuronal development, and the intimate communication between lymphocytes within the immune system . The polarization of yeast cells shares many features with that of these more complex examples, including regulation by both intrinsic and extrinsic cues, conserved regulatory molecules such as Cdc42 GTPase, and asymmetry of the cytoskeleton as its centerpiece . This review summarizes the molecular pathways governing the generation of cell polarity in yeast.

Mol Cell Biol, 2000 Jan, 20(2), 563 - 74
Feedback phosphorylation of the yeast a-factor receptor requires activation of the downstream signaling pathway from G protein through mitogen-activated protein kinase; Feng Y et al.; The two yeast pheromone receptors, the a and alpha-factor receptors, share many functional similarities: both G protein-coupled receptors couple to the same downstream signal transduction pathway, and both receptors undergo feedback regulation involving increased phosphorylation on their C-terminal domains in response to ligand challenge . The present work, which focuses on the signaling mechanism controlling this feedback phosphorylation, indicates one striking difference . While the alpha-factor-induced phosphorylation of the alpha-factor receptor does not require activation of the downstream G protein-directed signaling pathway (B . Zanolari, S . Raths, B . Singer-Kruger, and H . Riezman, Cell 71:755-763, 1992), the a-factor-induced phosphorylation of the a-factor receptor (Ste3p) clearly does . Induced Ste3p phosphorylation was blocked in cells with disruptions of various components of the pheromone response pathway, indicating a requirement of pathway components extending from the G protein down through the mitogen-activated protein kinase (MAPK) . Furthermore, Ste3p phosphorylation can be induced in the absence of the a-factor ligand when the signaling pathway is artificially activated, indicating that the liganded receptor is not required as a substrate for induced phosphorylation . While the activation of signaling is critical for the feedback phosphorylation of Ste3p, pheromone-induced gene transcription, one of the major outcomes of pheromone signaling, appears not to be required . This conclusion is indicated by three results . First, ste12Delta cells differ from cells with disruptions of the upstream signaling elements (e.g., ste4Delta, ste20Delta, ste5Delta, ste11Delta, ste7Delta, or fus3Delta kss1Delta cells) in that they clearly retain some capacity for inducing Ste3p phosphorylation . Second, while activated alleles of STE11 and STE12 induce a strong transcriptional response, they fail to induce a-factor receptor phosphorylation . Third, blocking of new pheromone-induced protein synthesis with cycloheximide fails to block phosphorylation . These findings are discussed within the context of a recently proposed model for pheromone signaling (P . M . Pryciak and F . A . Huntress, Genes Dev . 12:2684-2697, 1998): a key step of this model is the activation of the MAPK Fus3p through the G(betagamma)-dependent relocalization of the Ste5p-MAPK cascade to the plasma membrane . Ste3p phosphorylation may involve activated MAPK Fus3p feeding back upon plasma membrane targets.

Mol Cell Biol, 2000 Jan, 20(2), 488 - 95
A novel subunit of yeast RNA polymerase III interacts with the TFIIB-related domain of TFIIIB70; Ferri ML et al.; There is limited information on how eukaryotic RNA polymerases (Pol) recognize their cognate preinitiation complex . We have characterized a polypeptide copurifying with yeast Pol III . This protein, C17, was found to be homologous to a mammalian protein described as a hormone receptor . Deletion of the corresponding gene, RPC17, was lethal and its regulated extinction caused a selective defect in transcription of class III genes in vivo . Two-hybrid and coimmunoprecipitation experiments indicated that C17 interacts with two Pol III subunits, one of which, C31, is important for the initiation reaction . C17 also interacted with TFIIIB70, the TFIIB-related component of TFIIIB . The interaction domain was found to be in the N-terminal, TFIIB-like half of TFIIIB70, downstream of the zinc ribbon and first imperfect repeat . Although Pol II similarly interacts with TFIIB, it is notable that C17 has no similarity to any Pol II subunit . The data indicate that C17 is a novel specific subunit of Pol III which participates together with C34 in the recruitment of Pol III by the preinitiation complex.

Mol Cell Biol, 2000 Jan, 20(2), 478 - 87
CLN1 and its repression by Xbp1 are important for efficient sporulation in budding yeast; Mai B et al.; Xbp1, a transcriptional repressor of Saccharomyces cerevisiae with homology to Swi4 and Mbp1, is induced by stress and starvation during the mitotic cycle . It is also induced late in the meiotic cycle . Using RNA differential display, we find that genes encoding three cyclins (CLN1, CLN3, and CLB2), CYS3, and SMF2 are downregulated when Xbp1 is overexpressed and that Xbp1 can bind to sequences in their promoters . During meiosis, XBP1 is highly induced and its mRNA appears at the same time as DIT1 mRNA, but its expression remains high for up to 24 h . As such, it represents a new class of meiosis-specific genes . Xbp1-deficient cells are capable of forming viable gametes, although ascus formation is delayed by several hours . Furthermore, Xbp1 target genes are normally repressed late in meiosis, and loss of XBP1 results in their derepression . Interestingly, we find that a deletion of CLN1 also reduces the efficiency of sporulation and delays the meiotic program but that sporulation in a Deltacln1 Deltaxbp1 strain is not further delayed . Thus, CLN1 may be Xbp1's primary target in meiotic cells . We hypothesize that CLN1 plays a role early in the meiotic program but must be repressed, by Xbp1, at later stages to promote efficient sporulation.

Mol Cell Biol, 2000 Jan, 20(2), 441 - 52
Yeast exosome mutants accumulate 3'-extended polyadenylated forms of U4 small nuclear RNA and small nucleolar RNAs; van Hoof A et al.; The exosome is a protein complex consisting of a variety of 3'-to-5' exonucleases that functions both in 3'-to-5' trimming of rRNA precursors and in 3'-to-5' degradation of mRNA . To determine additional exosome functions, we examined the processing of a variety of RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P, RNase MRP, and SRP RNAs, and 5S rRNAs in mutants defective in either the core components of the exosome or in other proteins required for exosome function . These experiments led to three important conclusions . First, exosome mutants accumulate 3'-extended forms of the U4 snRNA and a wide variety of snoRNAs, including snoRNAs that are independently transcribed or intron derived . This finding suggests that the exosome functions in the 3' end processing of these species . Second, in exosome mutants, transcripts for U4 snRNA and independently transcribed snoRNAs accumulate as 3'-extended polyadenylated species, suggesting that the exosome is required to process these 3'-extended transcripts . Third, processing of 5.8S rRNA, snRNA, and snoRNA by the exosome is affected by mutations of the nuclear proteins Rrp6p and Mtr4p, whereas mRNA degradation by the exosome required Ski2p and was not affected by mutations in RRP6 or MTR4 . This finding suggests that the cytoplasmic and nuclear forms of the exosome represent two functionally different complexes involved in distinct 3'-to-5' processing and degradation reactions.

J Biol Chem, 1999 Dec 31, 274(53), 38119 - 24
Prenylation of Rho1p is required for activation of yeast 1, 3-beta-glucan synthase; Inoue SB et al.; One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeast Saccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1, 3-beta-glucan synthase . Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the beta subunit of geranylgeranyl transferase type I called cal1-1 . Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-beta-glucan synthase . The 1,3-beta-glucan synthase activity of the cal1-1 membrane was significantly reduced compared with that of the wild-type membrane . The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1, 3-beta-glucan synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p . Glutathione S-transferase (GST)-Rho1 proteins with or without the C-terminal motif required for the modification were purified and used to analyze the interaction . The modified form of GST-Rho1p was specifically able to restore the 1,3-beta-glucan synthase of the rho1-3 membrane . Gel overlay analysis indicated that an unmodified form of GST-Rho1p fails to interact with Fks1p . These results indicated that the geranylgeranylation of Rho1p is a prerequisite to the assembly and activation of 1,3-beta-glucan synthase in vitro . Increased cytoplasmic levels of divalent cations such as Ca(2+) restored both Rho1p modification and the 1,3-beta-glucan synthase activity of cal1-1, suggesting that cytoplasmic levels of the divalent cations affect geranylgeranyl transferase type I activity in vivo.

Curr Opin Microbiol, 1999 Dec, 2(6), 618 - 23
Yeast as a tool for apoptosis research; Matsuyama S et al.; Apoptosis is a unique cell suicide process that plays important roles in a wide variety of developmental and normal physiological processes in animal species, and causes diseases when inappropriately controlled . Although yeast do not possess the proteases ultimately responsible for the morphological events recognized as apoptosis, these simple unicellular eukaryotes can serve as a powerful tool for apoptosis researchers . Ectopic expression of several human and animal apoptosis proteins in either budding or fission yeast results in phenotypes that create opportunities for genetic screens . Recent exploitation of yeast as tools for studying human apoptosis-regulatory proteins has yielded novel insights into cell death mechanisms, suggesting strategies for identification of genes and drugs that modulate the functions of proteins involved in apoptosis control.

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