No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 100-124).

Thesis (PhD)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 143-156). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 30-37).

The bipolar budding pattern of a/a Saccharomyces cerevisiae cells appears to depend on persistent spatial markers. Genetic analysis reported here indicates that BUD8 and BUD9 potentially encode components of the markers at the distal and proximal poles, respectively. Mutants deleted for BUD8 or BUD9 bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. Both molecules are related plasma membrane glycoproteins that are both N- and O-glycosylated. Each protein was localized predominantly in the expected location, with Bud8p delivered to the presumptive bud site just before bud emergence, and Bud9p delivered to the bud side of the mother-bud neck just before cytokinesisis. Promoter-swap experiments revealed the importance of time of transcription in localization: expression of Bud8p from the BUD9 promoter leads to its localization predominantly in the sites typical for Bud9p, and vice versa. Moreover, expression of Bud8p from the BUD9 promoter fails to rescue the budding-pattern defect of a bud8 mutant but fully rescues that of a bud9 mutant. However, although expression of Bud9p from the BUD8 promoter fails to rescue a bud9 mutant, it also rescues only partially the budding-pattern defect of a bud8 mutant. / Using a collection of mutants individually deleted for almost every yeast gene, I undertook a genome-wide phenotype analysis for altered sensitivity to a yeast antifungal protein, the K1 killer toxin. Mutations in most genes have no effect on toxin sensitivity, with less than 10% having a phenotype. Only 4% of these were previously known to have a toxin phenotype. There is a markedly non-random functional distribution of mutants with a toxin phenotype. Many genes fall into a limited set of functional classes or modules, which define specific areas of cellular function. These include known pathways of cell wall synthesis and signal transduction, and offer new insights into these processes and into cell wall morphogenesis.

CWH41 is required for beta-1,6-glucan biosynthesis and encodes glucosidase I, an enzyme involved in protein N-chain glucose processing. Therefore, the effects of N-chain glucosylation and processing on beta-1,6-glucan biosynthesis were examined, and it was shown that incomplete N-chain glucose processing results in loss of beta-1,6-glucan. To explore the involvement of other N-chain-dependent events with beta-1,6-glucan synthesis, the S. cerevisiae KRE5 and CNE1 genes were investigated, which encode homologs of the 'quality control' components UDP-Glc:glycoprotein glucosyltransferase and calnexin, respectively. The essential activity of Kre5p was found to be separate from its possible role as a UDP-Glc:glycoprotein glucosyltransferase. A ∼30% decrease in beta-1,6-glucan was observed upon disruption of CNE1, a phenotype which is additive with other beta-1,6-glucan synthetic mutants. Analysis of the cell wall anchorage of alpha-agglutinin suggests the existence of two beta-1,6-glucan biosynthetic pathways, one N-chain dependent, the other involving protein glycosylphosphatidylinositol modification. / Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. The fks1Delta mutant was partial K1 killer toxin resistant and showed a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1Delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. FKS2 overexpression suppressed the killer toxin phenotype of fks1Delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1Delta cells. Eight out of twelve fks1tsfks2Delta mutants had altered beta-glucan levels at the permissive temperature: the FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive FKS1T6051 M761T allele increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. beta-1,6-Glucan deficient mutants had reduced in vitro glucan synthase activity and mislocalized Fks1p and Fks2p, possibly contributing to the observed beta-1,6-glucan defects.

We have previously demonstrated that Mid2p is required for the activation of the PKC1-MPK1 cell integrity pathway during cell exposure to mating pheromone, calcofluor white (CFW), and heat. Accumulating evidence indicates that Mid2p might regulate this pathway via the small GTPase, Rho1p. To understand the mechanism by which Mid2p signals, we initiated a two hybrid screen using the essential cytoplasmic tail of Mid2p as bait. ZEO1 (YOL109w), a previously uncharacterized open reading frame, was identified. ZEO1 encodes a 12kDa protein that co-localizes to the plasma membrane and interacts with the cytoplasmic tail of Mtl1p, a Mid2p functional homologue. Like mid2Delta mutants, cells deleted for ZEO1 are resistant to calcofluor white. In addition, ZEO1 null strains are no longer hypersensitive to calcofluor white caused by high copy expression of MID2. A role for Zeo1p in the cell integrity pathway is supported by the finding that disruption of ZEO1 leads to a Mid2p-dependent constitutive phosphorylation of Mpk1p. (Abstract shortened by UMI.)

Saccharomyces cerevisiae Kre5p is important for the biosynthesis of beta-1,6-glucan, which is required for proper cell wall assembly and architecture. A functional and cell biological analysis of Kre5p was conducted to further elucidate its role in beta-1,6-glucan synthesis. Kre5p was found to be a primarily soluble N-glycoprotein of ∼200 kD that localizes to the endoplasmic reticulum. Observation of Kre5p-deficient cells reveals a severe cell wall morphological defect, and kre5Delta cells were shown to have only residual levels of beta-1,6-glucan. KRE6 was identified as a multicopy suppressor of a temperature-sensitive kre5 allele, suggesting these proteins participate in a common pathway. An analysis of truncated versions of Kre5p indicates that it may have two independent, essential activities, or that it functions in a homodimeric state. Finally, Candida albicans KRE5 was shown to partially restore growth to kre5Delta cells, suggesting it has a function similar to that of the S. cerevisiae protein.

A search of the Saccharomyces genome data base revealed an open reading frame of 2039 amino acids with homology to L-type calcium channels. Northern blots probed with a 540 bp PCR product of the ORF showed a transcript of 6.1 kb. Two procedures were used to disrupt the gene: the ORF was truncated by an integrative disruption after the third pore motif, or deleted in the first three pore domains using a one-step disruption construct. In most strains tested, the disruptions gave no apparent phenotype when tested under a variety of conditions. However, conspicuous phenotypes were seen in the strain YEL161-2A, a strain super-sensitive to alpha-mating factor (sst1). In most respects, truncation gave a less severe phenotype than deletion, suggesting that the truncated gene retains partial function. Calcium uptake during normal growth, as well as the increased calcium uptake in response to mating factor, were reduced progressively by the truncation and deletion respectively. Growth rate and cell viability were reduced, cell size heterogeneity increased, and recovery from mating factor arrest was delayed and abnormal. The cells became sensitive to MnCl$ sb2.$ The phenotype resulting from gene truncation was alleviated by a high-calcium medium, and exacerbated by low calcium. Complementation of the deleted strain by a Yep13 plasmid containing BAR1 (SST1) restored normal growth and viability. However, somewhat paradoxically, deletion of the putative calcium channel gene in another sst1 strain (SY1159) showed no phenotype.