The Gas1 glycoprotein, a putative wall polymer cross-linker.

The yeast cell wall, which for years has been regarded as a static cellular component, has been revealed to be dynamic in its structure and composition and complex in its enzymatic activity. The S. cerevisiae cell wall is composed of beta-1,3/beta-1,6-glucans, mannoproteins, and chitin, which are assembled into an extracellular matrix essential for maintenance of cell integrity. Gas1p, a glycoprotein anchored to the outer leaflet of the plasma membrane through a glycosylphosphatidylinositol, plays a key role in cell wall assembly. Loss of Gas1p leads to several morphogenetic defects and to a decrease in the amount of cross-links between the cell wall glucans. These defects in turn trigger a compensatory response that guarantees cell viability. Several Gas1p homologs have been isolated from Candida species and S. pombe. The Gas1p family also includes two plant proteins with endo-beta-1,3-glucanase activity. Sequence comparisons reveal that Gas1p family proteins have a modular organization of domains. The genetic and molecular analyses reviewed here suggest that Gas1p could play a role as a polymer cross-linker, presumably by catalyzing a transglycosylation reaction.

Reduction of ribosome activity and synthesis of stable RNA in Neurospora crassa.

The addition of cycloheximide (0.02 micrograms/ml) to exponentially growing cultures of Neurospora crassa causes a reduction in growth rate and a decrease in the rate of protein accumulation, due to a partial inhibition of protein synthesis, while RNA accumulation is unaffected for about 1 h. Thus, an increased RNA:protein ratio is established in the presence of the inhibitor. RNA that accumulates during treatment with cycloheximide has the same characteristics as that of the control cultures and this, together with the enhancement of the relative rate of synthesis of ribosomal proteins induced by cycloheximide, seems to indicate that more mature ribosomes are present in cycloheximide-treated cultures. The endocellular level of several amino acids begins to increase significantly only 60 min after cycloheximide addition. A possible explanation of the stimulation of ribosome production induced by cycloheximide is given in terms of the existence of a feed-back mechanism controlling ribosome synthesis.

Cloning, sequencing and regulation of a cDNA encoding a small heat-shock protein from Schizosaccharomyces pombe.

We have isolated a Schizosaccharomyces pombe cDNA encoding a small heat-shock protein, designated Hsp9. The deduced amino acid sequence shares significant homology with the Saccharomyces cerevisiae Hsp12 gene product. Northern blot analysis identified a 600-base transcript which is expressed at a low level in S. pombe exponentially growing cells, but is strongly induced by heat-shock and upon entry into the stationary phase. An increase in the transcript level is also observed in response to glucose deprivation.

The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol.

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.

Immunochemical characterization of gp115, a yeast glycoprotein modulated by the cell cycle.

A cell cycle-modulated glycoprotein (gp115, 115 kDa, isoelectric point 4.8-5) of Saccharomyces cerevisiae has been purified by Concanavalin A-affinity chromatography, followed by preparative two-dimensional gel electrophoresis, from yeast membrane proteins solubilized in Triton X-100. Antisera have been generated against the electrophoretically purified protein. Their specificity has been established by immunoblot analysis and by comparison of the partial proteolytic map obtained for the immunoprecipitated 35S-labeled 115 kDa polypeptide with that of the in vivo [35S]methionine-labeled gp115 isolated from two-dimensional gels. In tunicamycin-treated cells the immunoblot analysis identifies an unglycosylated precursor (86-88 kDa) and in sec18 mutant cells at the restrictive temperature an intermediary precursor of about 100 kDa. Six to seven carbohydrate chains have been estimated to be present on the gp115 protein, accounting for an electrophoretic shift corresponding to about 27 to 29 kDa of its relative molecular mass. Affinity-purified antibodies against the unglycosylated precursor (86-88 kDa) of gp115 were prepared and used to localize gp115 by indirect immunofluorescence microscopy. The similarity between the pattern of fluorescence obtained with these antibodies and that obtained using anti-plasma membrane H+-ATPase antibodies suggests an association of gp115 with the plasma membrane.

Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway.

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2Delta mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of approximately 11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.

Immunological cross-reactivity of fungal and yeast plasma membrane H+-ATPase.

The plasma membrane H+-ATPases from fungi and yeasts have similar catalytic and molecular properties. A structural comparison has been performed using immunoblot analysis with polyclonal antibodies directed toward the 102 kDa polypeptide of the plasma membrane H+-ATPase from Neurospora crassa. A strong cross-reactivity is observed between the fungal H+-ATPase and the enzyme from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Structural homologies are indicated also by the analysis of the cross-reactive peptides originated by proteolytic digestion of Neurospora and S. cerevisiae purified enzymes. Neither enzyme from these two sources appears to be glycosylated by a highly sensitive concanavalin A affinity assay on blotted proteins. A glycoprotein of Mr 115000 and pI 4.8-5, which comigrates with a cell cycle-modulated protein on 2D gel, is present in partially purified preparations of plasma membrane H+-ATPase of S. cerevisiae and it is shown to be structurally unrelated to H+-ATPase.

Defects in assembly of the extracellular matrix are responsible for altered morphogenesis of a Candida albicans phr1 mutant.

Analysis of Candida albicans cells using antibodies directed against Gas1p/Ggp1p, Saccharomyces cerevisiae homolog of Phr1p, revealed that Phr1p is a glycoprotein of about 88 kDa whose accumulation increases with the rise of external pH. This polypeptide is present both in the yeast form and during germ tube induction. In the Phr1- cells at pH 8 the solubility of glucans in alkali is greatly affected. In the parental strain the alkali-soluble/-insoluble glucan ratio shows a 50% decrease at pH 8 with respect to pH 4.5, whereas in the null mutant it is unchanged, indicating the lack of a polymer cross-linker activity induced by the rise of pH. The mutant has a sixfold increase in chitin level and is hypersensitive to calcofluor. Consistently with a role of chitin in strengthening the cell wall, Phr1- cells are more sensitive to nikkomycin Z than the parental strain.

Identification of a labile protein involved in the G1-to-S transition in Saccharomyces cerevisiae.

The biochemical nature of the start process that commits budding yeast to DNA synthesis is not known. Kinetic evidence has suggested recently that short-lived protein(s) may have to accumulate to a critical level before the cell cycle may progress towards DNA synthesis and cell division. We investigated by high-resolution two-dimensional electrophoresis whether, in a cdc25-1 mutant strain of Saccharomyces cerevisiae that had been blocked at the regulatory step called "start" by growth at a restrictive temperature, short-lived proteins are synthesized during the recovery of growth at a permissive temperature. Of the approximately equal to 500 proteins resolved by the two-dimensional electrophoresis, 6 were short-lived. Only one of them (Mr = 100,000, pI approximately equal to 4.8-5) appears to be specifically made during the G1-to-S transition at start. A regulatory role for cell cycle progression in yeast is suggested for this protein, p100.

Control of the yeast cell cycle by protein synthesis.

The increased synthesis of ribosomal RNA (rRNA) is correlated with enhanced cell proliferation, and it has been suggested that rRNA metabolism may have a regulatory role in the progression of the cell cycle. Alternatively, it might be the ensuing more active protein synthesis that drives the cell cycle progression. We have found that treatment with low doses of cycloheximide dissociates rRNA and protein synthesis. In fact, after the addition of cycloheximide the protein synthesis rate is strongly inhibited, whereas the rate of rRNA synthesis is unaffected for some time. The progression of the cell cycle, monitored as analysis of DNA distribution by flow cytometry and as bud emergence, is quickly and largely inhibited, thus indicating that a sustained rRNA metabolism is not sufficient to allow continuous cycle progression. The effects of cycloheximide on the daughter and mother duplication times, on the mean cell volume, and on the volume at budding were also analyzed. The results suggest that protein synthesis, rather than rRNA synthesis, may have a key role in the control of cell cycle progression in Saccharomyces cerevisiae.

Identification of a glycoprotein involved in cell cycle progression in yeast.

The molecular events of start, the regulatory step that commits yeast cells to DNA replication, have recently begun to be investigated. One of the gene products required for completion of start has been found to have a significant structural homology with oncogenes endowed with protein kinase activity. Our experiments provide data on the biosynthetic pathway of a previously identified labile protein (p100, molecular weight 100,000, isoelectric point of approximately 4.8-5) involved in cell cycle progression at start, which appears to be specifically made during the release from cell cycle arrest of a temperature-sensitive mutant (cdc25) of Saccharomyces cerevisiae. On two-dimensional gel, p100 migrates very close to another 100-kDa labile protein (p100*) which behaves as a cell cycle modulated protein with reduced synthesis in G1. Pulse and chase labeling of protein with [35S]methionine suggests that both p100 and p100* are processed to a protein (p115) of slightly higher molecular weight (Mr = 115,000). Peptide mapping analysis indicates that p100 and p100 yield identical maps and that both p100 and p100* are very much similar to p115. p115 is a glycosylated protein as shown by a labeling experiment with [3H]glucosamine and by the fact that the synthesis of both p100 and p115 is inhibited if cells are cultured in the presence of tunicamycin. A protein having the same heterogeneous aspect of migration on sodium dodecyl sulfate-polyacrylamide gel and the same apparent molecular weight and isoelectric point of p115 is abundantly present in a preparation of membranes from S. cerevisiae and the isolated radioactive p115 comigrates with it. Taken together these results favor the idea that terminal glycosylation of both p100 and p100* gives rise to the fully glycosylated p115 protein which appears to be a membrane-associated protein.

The yeast cell-wall salvage pathway.

The integrity of the cell wall depends on the synthesis and correct assembly of its individual components. Several environmental factors, such as temperature up-shift, treatments with mating factors or with specific cell wall-perturbing drugs, or genetic factors, such as inactivation of cell wall-related genes (for example FKS1 or GAS1) can impair construction of the cell wall. As the cell wall is essential for preserving the osmotic integrity of the cell, several responses are triggered in response to cell-wall damage. This review focuses on the activation of salvage pathways that guarantee cell survival through remodeling of the extracellular matrix. These researches have useful implication for the study of similar pathways in human fungal pathogens, and for the evaluation of the efficacy of new antifungal drugs.

Effect of tunicamycin on cell cycle progression in budding yeast.

Tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharides, prevents bud formation and growth in Saccharomyces cerevisiae cells that are either growing exponentially or recovering from different cell cycle arrests at start. Analysis of tunicamycin-treated cells by flow microfluorometry clearly shows that cells have a postsynthetic DNA content, but there is no evidence of an increase in binucleate cells. Therefore tunicamycin affects bud emergence and initiation of DNA synthesis, two events correlated under physiological conditions, in different ways. A bulk glycoprotein synthesis is shown to be required for bud emergence and localized chitin deposition, probably to sustain directional secretory vesicle transport, which allows polar growth of the bud. No evidence for a glycoprotein requirement for entrance into the S phase is obtained from the present experiments.

Increase of external osmolarity reduces morphogenetic defects and accumulation of chitin in a gas1 mutant of Saccharomyces cerevisiae.

We have performed a physiological analysis of the effects of high-osmolarity media on gas1Delta cells. The reductions in the duplication time, number of pluribudded cells, hypersensitivity to Calcofluor and sodium dodecyl sulfate, and chitin level indicate a partial suppression of the mutant phenotype. GAS1 deletion was found to be lethal in the absence of the Bck1 and Slt2 (Mpk1) proteins of the cell integrity pathway.

Transcript accumulation of the GGP1 gene, encoding a yeast GPI-anchored glycoprotein, is inhibited during arrest in the G1 phase and during sporulation.

The GGP1 (GAS1) gene encodes an exocellular 115-kDa glycoprotein (gp115) of the yeast Saccharomyces cerevisiae. We have monitored the changes in GGP1 mRNA levels under different conditions of G1 arrest. Transcript levels rapidly decrease during transition from exponential growth to stationary phase. They also decrease in the ts cdc25 and cdc28 START mutants when brought to the restrictive temperature. In cells arrested in G1 by alpha F treatment, the GPP1 mRNA level undergoes a threefold reduction. During release from the G1 block the mRNA level rapidly increases with a maximum at the onset of budding. During sporulation GGP1 mRNA level steadily decreases. These results indicate that the accumulation of the GGP1 transcript is inhibited during arrest in the G1 phase and during entry into the differentiative pathway of meiosis and sporulation. The induction of expression upon entry into the mitotic cycle suggests that GGP1 could be one of the genes whose transcription is activated at START.

Expression and secretion of beta-galactosidase in Saccharomyces cerevisiae using the signal sequences of GgpI, the major yeast glycosylphosphatidylinositol-containing protein.

New secretory signals and strategies can be attempted to improve the secretion of heterologous proteins of biotechnological interest which encounter difficulties being exported in yeast. The GGPI gene of Saccharomyces cerevisiae codes for a 125 kDa glycoprotein transported through the secretory pathway and anchored to the plasma membrane by means of a glycosylphosphatidylinositol. The regions coding for the secretory signal or also for the first 46 amino acids were tested for efficiency in secretion by fusion to the lacZ gene of Escherichia coli resulting in the synthesis of the endoplasmic reticulum-targeted 1-22- and 1-68-GgpIp/beta-gal hybrids. A cytoplasmic form was also examined. The 1-22 beta gal is partially transported to the cell surface and in the medium in an unglycosylated form. The 1-68 beta gal is completely retained in the intracellular membranes and is N-glycosylated in the GgpIp moiety. The amount of hybrid protein produced is similar and independent from its targeted site, suggesting that translocation through endoplasmic reticulum is not a limiting step, whereas the amount of active enzyme is from 50 to 80% lower for the endoplasmic reticulum forms compared with the cytoplasmic form. BiP/Kar2p putative precursor is accumulated in cells expressing the endoplasmic reticulum-targeted forms but not in those producing the cytosolic beta-galactosidase or over-expressing an endogenous secretory protein. Thus, glycosylation and abnormal folding rather than over-expression are among the factors responsible for the decreased activity and exit of beta-galactosidase from the endoplasmic reticulum and for induction of BiP. The results obtained indicate that the sole secretory signal of GgpIp is suitable to drive secretion of foreign products with complex folding and point to the importance of the endoplasmic reticulum quality control in the secretion of heterologous proteins in yeast.

Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.

Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.

Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.

cAMP promotes the synthesis in early G1 of gp115, a yeast glycoprotein containing glycosyl-phosphatidylinositol.

The glycoprotein gp115 (Mr = 115,000, pI 4.8-5) is localized in the plasma membrane of Saccharomyces cerevisiae cells and maximally expressed during G1 phase. To gain insight on the mechanism regulating its synthesis, we have examined various conditions of cell proliferation arrest. We used pulse-labeling experiments with [35S]methionine and two-dimensional gel electrophoresis analysis, which allow the detection of the well characterized 100-kDa precursor of gp115 (p100). In the cAMP-requiring mutant cyr1, p100 synthesis is active during exponential growth, shut off by cAMP removal, and induced when growth is restored by cAMP readdition. The inhibition of p100 synthesis also occurs in TS1 mutant cells (ras1ras2-ts1) shifted from 24 to 37 degrees C. During nitrogen starvation of rca1 cells, a mutant permeable to cAMP, p100 synthesis is also inhibited. cAMP complements the effect of ammonium deprivation, promoting p100 synthesis, even when added to cells which have already entered G0. Experiments with the bcy1 and cyr1bcy1 mutants have indicated the involvement of the cAMP-dependent protein kinases in the control of p100 synthesis. Moreover, the synthesis of p100 was unaffected in A364A cells, terminally arrested at START B by alpha-factor. These results indicate that the switch operating on p100 synthesis is localized in early G1 (START A) and is one of the multiple events controlled by the cAMP pathway.

Physiological analysis of mutants indicates involvement of the Saccharomyces cerevisiae GPI-anchored protein gp115 in morphogenesis and cell separation.

This paper reports a phenotypic characterization of ggp1 mutants. The cloned GGP1 (GAS1) gene, which encodes a major GPI-anchored glycoprotein (gp115) of Saccharomyces cerevisiae of unknown function, was used to direct the inactivation of the chromosomal gene in haploid and diploid strains by gene replacement. The analysis of the null mutants reveals a reduction in the growth rate of 15 to 40%. Cells are round, with more than one bud, and extensively vacuolized. In the stationary phase, mutant cells are very large, arrest with a high percentage of budded cells (about 54 and 70% for haploid and diploid null mutants, respectively, in comparison with about 10 to 13% for control cells), and have reduced viability. The observed phenotype suggests defects in cell separation. Flow cytometric analysis of DNA reveals an increase in the fraction of cells in the G2+M+G1* compartment during exponential growth. Conjugation and sporulation are not affected. The exocellular location of gp115 led us to examine cell wall properties. Cell wall and septum ultrastructure of abnormally budded cells was analyzed by electron microscopy analysis, and no appreciable differences from wild-type cells were found. Microscopic analysis revealed an increase in chitin content and delocalization. In comparison with control cells, ggp1 null mutants are shown to be resistant to Zymolyase during the exponential growth phase. A fivefold overexpression of gp115 does not bring about any effects on cell growth parameters and cell wall properties.