Seven temperature-sensitive cell lysis (cy) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY1S gene was isolated by complementation of the clyl5 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. 0. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990 The yeast cell wall is a complex rigid structure, responsible for cell shape, which undergoes a series of significant changes during the mitotic cell cycle (6). For example, bud emergence requires modifications of the cell wall at a precisely localized site and controlled cell wall growth in order to produce a daughter cell. Other cellular processes such as mating and sporulation also involve specific alterations of the cell wall structure.Yeast cell walls are constructed almost entirely of two classes of polysaccharides: polymers of mannose covalently linked to peptides, which are termed mannoproteins, and polymers of glucose, termed glucans. A third sugar polymer of N-acetylglucosamine, chitin, is present in only minor amounts (8). A number of Saccharomyces cerevisiae mutants with defects in the synthesis of cell wall components have been isolated. Defects in agglutination with a 1,3-alinked mannose antiserum define mannoprotein (mnn) mutants (reviewed in Ballou [1]), yeast killer toxin-resistant (kre) mutants display reduced levels of 1,6-,-glucan in the cell wall (2), and chitin synthase mutants are unable to convert exogenous glucosamine to chitin (3,22). The major cell wall-degrading enzyme activities mainly consist of 1,3-,-and

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta-glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.

We have previously described a temperature-sensitive pmi40-1 mutant of Saccharomyces cerevisiae which is defective in glycosylation and secretion because of a thermolabile phosphomannose isomerase (PMI) activity.Inactivation of PMI at the restrictive temperature of 37°C prevents synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death. Here, we report the isolation of the PMI40 gene by complementation of the corresponding mutation. The PMI40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S. cerevisiae in that it is short and the branch-forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC. The 48.2-kDa protein predicted to be encoded by PMI40 contains amino acid sequences corresponding to those of internal peptides derived from purified S. cerevisiae PMI. Deletion of the PMI40 coding sequence results in a strain requiring D-mannose for growth. The PMI40 gene is located on chromosome V, and its transcription is increased 12-fold when cells are grown on D-mannose as sole carbon source instead of D-glucose. PMI enzyme activity, however, is not increased in D-mannose-grown cells, and PMI protein levels remain constant, suggesting that the PMI40 gene is subject to additional levels of regulation. Dolichol-phosphate-mannose (Dol-P-Man) and GDP-mannose (GDP-Man) serve as the mannose donors for a number of important mannosylation reactions found in eukaryotes. These include formation of the mannosyl residues found in glycoproteins, where the mannosyl residues can be attached to both serine/threonine (0-linked) and asparagine (Nlinked) (9, 24, 33, 34) yeast mannosyl-and inositol-containing sphingolipids (1), and the glycosyl phosphatidylinositol moiety found linked to certain membrane proteins (5,8,18,23). The pathway of GDP-Man and Dol-P-Man synthesis and many of the glycosylation reactions in which these molecules participate are similar in higher and lower eukaryotes, and Saccharomyces cerevisiae has provided a useful model system for the study of glycosylation (la, 17). Indeed, it has been shown that the S. cerevisiae Dol-P-Man synthase DPM1 gene (24) can complement a glycosylation-defective mammalian cell line (la).The first step in the synthesis of GDP-Man and Dol-P-Man from glycolytic intermediates is performed by phosphomannose isomerase (EC 5.3.1.8), which catalyzes the reversible isomerization of fructose-6-phosphate and mannose-6-phosphate (13) (Fig. 1). We have previously described a temperature-sensitive lethal mutant of S. cerevisiae which possesses a thermolabile phosphomannose isomerase (PMI) activity due to the pmi40-1 mutation (26). At the restrictive temperature of 37°C, strains carrying pmi40-1 display an abnormal clumped morphology, are unable to secrete extracellular glycoproteins, produce cell walls deficient in D-mannose, and undergo cell lysis as measured by the release of intracellular enzymes. All of these defects...

The Saccharomyces cerevisiae HIS7 gene was cloned by its location immediately downstream of the previously isolated and characterized ARO4 gene. The two genes have the same orientation with a distance of only 416 bp between the two open reading frames. The yeast HIS7 gene represents the first isolated eukaryotic gene encoding the enzymatic activities which catalyze the fifth and sixth step in histidine biosynthesis. The open reading frame of the HIS7 gene has a length of 1,656 bp resulting in a gene product of 552 amino acids with a calculated molecular weight of 61,082. Two findings implicate a bifunctional nature of the HIS7 gene product. First, the N-terminal and C-terminal segments of the deduced HIS7 amino acid sequence show significant homology to prokaryotic monofunctional glutamine amidotransferases and cyclases, respectively, involved in histidine biosynthesis. Second, the yeast HIS7 gene is able to suppress His auxotrophy of corresponding Escherichia coli hisH and hisF mutants. HIS7 gene expression is regulated by the general control system of amino acid biosynthesis. GCN4-dependent and GCN4-independent (basal) transcription use different initiator elements in the HIS7 promoter.