Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae.

To further our studies of protein sorting and biogenesis of the lysosome-like vacuole in yeast, we have isolated spontaneous mutations in 11 new VPL complementation groups, as well as additional alleles of the eight previously described VPL genes. These mutants were identified by selecting for cells that mislocalize vacuolar proteins to the cell surface. Morphological examination of the vpl mutants indicated that most contain vacuoles of normal appearance; however, some of the mutants generally lack a large vacuole, and instead accumulate smaller organelles. Of the 19 VPL complementation groups, 12 were found to be identical to 12 of 33 VPT complementation groups identified in a separate study. Moreover, the end1 mutant and all of the previously reported pep mutants, with the exception of pep4, were found to exhibit a profound vacuolar protein sorting defect, and complementation tests between the PEP, VPL VPT and END1 groups demonstrated that there are extensive overlaps between these groups. Collectively, mutants in these four collections define 49 complementation groups required to deliver or retain soluble vacuolar enzymes, including carboxypeptidase Y (CPY) and proteinase A. We have also isolated 462 new mutants that lack normal levels of vacuolar CPY activity. Among these latter mutants, only pep4 mutants were found to be specifically defective in vacuolar zymogen activation. We conclude that there is a large number of gene products required for sorting or retention of vacuolar proteins in yeast, and only a single gene, PEP4, that is essential for activation of CPY and other vacuolar zymogens.     

Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae

The trehalose-degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen-starved glucose-repressed yeast (Saccharomyces cerevisiae) cells. Trehalase activation is mediated by phosphorylation. Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles. Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro. To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22. Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source. Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive. After the addition of glucose or of a nitrogen source to starved cells, Pph22-overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress-regulated element-controlled genes. Deletion of the SCH9 gene encoding a protein kinase involved in nutrient-induced signal transduction restored glucose-induced trehalase activation in Pph22-overexpressing cells. Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient-induced signal transduction pathways in the absence of nutrients.     

Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.     

Stationary phase in the yeast Saccharomyces cerevisiae.

Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion, (vi) membrane biosynthesis, and (vii) cell polarity. The exact nature of the relationship between these processes and survival during stationary phase remains to be elucidated. We propose that cell cycle arrest coordinated with the ability to remain viable in the absence of additional nutrients provides a good operational definition of starvation-induced stationary phase.     

Characterization of an estrogen-binding protein in the yeast Saccharomyces cerevisiae

This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae. [3H]Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns. Other 3H-steroids did not exhibit specific binding. [3H]Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding. The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol. Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for [3H]estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein. Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A. Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation. The estrogen binder in S. cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus. 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones. In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S. cerevisiae. We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule.     

Tandemly arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces cerevisiae.

Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA. These genes are arranged in a single tandem array of 100 repeats. Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene. Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA. The coding sequence of this gene is different from that of the "normal" 5S gene in three positions located at the 3' end of the gene.     

Ascospore formation in the yeast Saccharomyces cerevisiae.

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae.

The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.     

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins.

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.     

Localization of a 210-kDa microtubule-interacting protein in the yeast Saccharomyces cerevisiae.

Using the monoclonal antibody MA-01, which recognizes a 210-kDa protein in cell-free extracts, spindle and cytoplasmic microtubules were visualized in budding yeast, Saccharomyces cerevisiae. In additional, a spot-like staining was found beneath the plasma membrane, revealing in part correlation with F-actin distribution. This pattern was common for cells of all cell-cycle stages. The interaction of the protein recognized by MA-01 with microtubules was confirmed in the double labeling with a polyclonal antitubulin antibody and by the sensitivity of intranuclear structures stained by MA-01 to the microtubule disrupting drug nocodazole.     

Iron-reductases in the yeast Saccharomyces cerevisiae

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.     

Multiple genes encode the translation elongation factor EF-1 gamma in Saccharomyces cerevisiae.

A gene encoding a yeast homologue of translation elongation factor 1 gamma (EF-1 gamma), TEF3, was isolated as a gene dosage extragenic suppressor of the cold-sensitive phenotype of the Saccharomyces cerevisiae drs2 mutant. The drs2 mutant is deficient in the assembly of 40S ribosomal subunits. We have identified a second gene, TEF4, that encodes a protein highly related to both the Tef3p protein (Tef3p), and EF-1 gamma isolated from other organisms. In contrast to TEF3, the TEF4 gene contains an intron. Gene disruptions showed that neither gene is required for mitotic growth. Haploid spores containing disruptions of both genes are viable and have no defects in ribosomal subunit composition or polyribosomes. Unlike TEF3, extra copies of TEF4 do not suppress the cold-sensitive 40S ribosomal subunit deficiency of a drs2 strain. Low-stringency genomic Southern hybridization analysis indicates there may be additional yeast genes related to TEF3 and TEF4.     

New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae.

A mutation causing resistance to carbon catabolite repression in gene HEX2, mutant allele hex2-3, causes an extreme sensitivity to maltose when in combination with the genes necessary for maltose metabolism. This provided a convenient system for the selective isolation of mutations in genes specifically required for maltose metabolism and other genes involved in general carbon catabolite repression. In addition to reversion of the hex2-3 allele, mutations in three other genes were detected. These genes were called CAT1, CAT3, and MUR1 and in a mutated form abolished maltose inhibition caused by mutant allele hex2-3. Mutant alleles cat1 and cat3 also restored normal repression in the presence of the hex2-3 allele. Segregants having only mutant alleles cat1 or cat3 were obtained by tetrad analysis. These segregants could not grow on nonfermentable carbon sources. Mutant alleles of gene CAT1 were allelic to a mutant allele cat1-1 previously isolated (Zimmermann et al., Mol. Gen. Genet. 151:95-103). Such mutants prevented derepression not only of the maltose catabolizing system, the selected property, but also of glyoxylate shunt and gluconeogenic enzymes. However, respiratory activities and invertase formation were not affected under derepressing conditions. cat3 mutants had the same phenotypic properties as cat1 mutants. This showed that carbon metabolism in yeast cells is under a very complex and ramified control of repressing and derepressing genes, which are interdependent.     

Aberrant protein phosphorylation at tyrosine is responsible for the growth-inhibitory action of pp60v-src expressed in the yeast Saccharomyces cerevisiae.

Expression of pp60v-src, the transforming protein of Rous sarcoma virus, arrests the growth of the yeast Saccharomyces cerevisiae. To determine the basis of this growth arrest, yeast strains were constructed that expressed either wild-type v-src or various mutant v-src genes under the control of the galactose-inducible, glucose repressible GAL1 promoter. When shifted to galactose medium, cells expressing wild-type v-src ceased growth immediately and lost viability, whereas cells expressing a catalytically inactive mutant (K295M) continued to grow normally, indicating that the kinase activity of pp60v-src is required for its growth inhibitory effect. Mutants of v-src altered in the SH2/SH3 domain (XD4, XD6, SPX1, and SHX13) and a mutant lacking a functional N-terminal myristoylation signal (MM4) caused only a partial inhibition of growth, indicating that complete growth inhibition requires either targeting of the active kinase or binding of the kinase to phosphorylated substrates, or both. Cells arrested by v-src expression displayed aberrant microtubule structures, alterations in DNA content and elevated p34CDC28 kinase activity. Immunoblotting with antiphosphotyrosine antibody showed that many yeast proteins, including the p34CDC28 kinase, became phosphorylated at tyrosine in cells expressing v-src. Both the growth inhibition and the tyrosine-specific protein phosphorylation observed following v-src expression were reversed by co-expression of a mammalian phosphotyrosine-specific phosphoprotein phosphatase (PTP1B). However a v-src mutant with a small insertion in the catalytic domain (SRX5) had the same lethal effect as wild-type v-src, yet induced only very low levels of protein-tyrosine phosphorylation. These results indicate that inappropriate phosphorylation at tyrosine is the primary cause of the lethal effect of pp60v-src expression but suggest that only a limited subset of the phosphorylated proteins are involved in this effect.     

Formamidopyrimidine DNA glycosylase in the yeast Saccharomyces cerevisiae.

A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.     

Osmotic mass transfer in the yeast Saccharomyces cerevisiae.

This paper reviews the passive mechanisms involved in the response of a yeast to changes in medium concentration and osmotic pressure. The results presented here were collected in our laboratory during the last decade and are experimentally based on the measurement of cell volume variations in response to changes in the medium composition. In the presence of isoosmotic concentration gradients of solutes between intracellular and extracellular media, mass transfers were found to be governed by the diffusion rate of the solutes through the cell membrane and were achieved within a few seconds. In the presence of osmotic gradients, mass transfers mainly consisting in a water flow were found to be rate limited by the mixing systems used to generate a change in the medium osmotic pressure. The use of ultra-rapid mixing systems allowed us to show that yeast cells respond to osmotic upshifts within a few milliseconds and to determine a very high hydraulic permeability for yeast membrane (Lp>6.10(-11) m x sec)-1) x Pa(-1)). This value suggested that yeast membrane may contain facilitators for water transfers between intra and extracellular media, i.e. aquaporins. Cell volume variation in response to osmotic gradients was only observed for osmotic gradients that exceeded the cell turgor pressure and the maximum cell volume decrease, observed during an hyperosmotic stress, corresponded to 60% of the initial yeast volume. These results showed that yeast membrane is highly permeable to water and that an important fraction of the intracellular content was rapidly transferred between intracellular and extracellular media in order to restore water balance after hyperosmotic stresses. Mechanisms implied in cell death resulting from these stresses are then discussed.