The Mpt5 protein of Saccharomyces cerevisiae has been implicated in a variety of cellular processes including cell cycle regulation, aging, cell wall maintenance and regulation of gene transcription by glucose. Loss of Mpt5p results in temperature-sensitive cell lysis which is osmotically remedial. In this thesis, synthetic lethalities of the mpt5Delta msn5Delta double mutant and the cln1Delta cln2Deltampt5Delta triple mutant are reported. A multicopy suppressor screen identified amino-terminal truncations of Mpt5p and the HMLalpha silent mating type locus as dosage-dependent suppressors of the mpt5Delta is lethality. Differential gene expression analysis in mpt5Delta revealed that several genes which code for cell wall structural proteins and genes which are under control of the Tup1p-repressor are less expressed in the absence of Mpt5p. Finally, Green Fluorescent Protein-tagged Mpt5p has allowed for investigation of Mpt5p cellular localization.

Two assay systems were designed and constructed in this study in order to investigate mRNA secondary structure melting during translation elongation in <i>S. cerevisiae</i>. The first, where stable secondary structures were placed within a <i>lacZ</i> reporter mRNA, was used to demonstrate that such structures are resolved efficiently, as the insertion of a stable stem-loop of -66 kcal/mol into the <i>lacZ</i> coding region only reduced translation efficiency by 40%. It was further demonstrated that the effect of secondary structures in open reading frames are not additive, as two stem-loops of -45 kcal/mol adjacent to one another did not decrease translation efficiency, to the extent expected of a single stem-loop of -90 kcal/mol. The L-A d.s. RNA virus RNA pseudoknot had no effect on translation elongation. A second assay, based on -1 frameshifting upstream of RNA secondary structure elements was used to test candidate genes for a role in mRNA secondary structure melting during translation elongation. It was demonstrated that two cytoplasmic RNA helicases, Ded1p and Dbp5p are not involved in this process. In contrast to previous work (Cui, <i>et. al</i>., 1996; EMBO 15:5726-5736) this study revealed that Upf1p, an RNA helicase involved in nonsense-mediated decay, played no role in -1 ribosomal frameshifting. Environmental effects on mRNA secondary structure melting were investigated by measuring secondary structure melting at 18^oC, using the -1 frameshift assay. Contrary to expectations, -1 frameshifting was not enhanced by low temperature, perhaps because a helicase-like activity is induced at 18^oC. Proteomic analysis revealed that increased levels of Gdh1p are associated with polyribosomes at 18^oC compared to 37^oC. Gdh1p interacts with Lsm1p, a component of an mRNA decapping complex, in a two-hybrid screen.

The Saccharomyces cerevisiae genes KRE6 and SKN1 encode a novel pair of highly homologous proteins involved in cell wall (1$ rightarrow$6)-$ beta$-glucan assembly. Disruption of KRE6 results in a slow growing, killer-toxin resistant mutant possessing reduced levels of structurally wild type (1$ rightarrow$6)-$ beta$-glucan. Although deletion of SKN1 has no effect on killer sensitivity, growth, or (1$ rightarrow$6)-$ beta$-glucan levels, SKN1 appears to overlap in function with KRE6, suppressing kre6 null alleles in a dosage-dependent manner. Strains deleted of both KRE6 and SKN1 possess an exaggerated growth phenotype, enhanced cell wall ultrastructure defects, and more severe (1$ rightarrow$6)-$ beta$-glucan reductions compared with either single disruptant. Moreover, the residual (1$ rightarrow$6)-$ beta$-glucan polymer in kre6 skn1 double mutants is smaller in size and altered in structure. Since single disruptions of either gene lead to structurally wild type (1$ rightarrow$6)-$ beta$-glucan, KRE6 and SKN1 appear to function independently and to act early in the assembly of the polymer, possibly as glucan synthases. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess C-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. / An initial characterization of Kre6p and Skn1p reveal both to be phosphorylated integral-membrane glycoproteins, with Kre6p likely localized to the Golgi apparatus. The topology implied by the post-translational modifications of Kre6p and Skn1p, offers the potential for both proteins to link cytoplasmic regulation with a secretory pathway-based assembly of the (1$ rightarrow$6)-$ beta$-glucan polymer. The observed phosphorylation of both Kre6p and Skn1p prompted an examination for genetic interactions with suspected cell wall regulating kinases. KRE6-dependent suppression of the pkc1 lysis defect, as well as synthetic lethal interactions between several KRE genes and members of the PKC1-mediated MAP kinase pathway, supports a role for the PKC1 pathway in regulating synthesis of cell wall components, including (1$ rightarrow$6)-$ beta$-glucan.

Analyses of genes involved in yeast cell wall $ beta$1,6-glucan assembly have identified CWH41, PTC1/CWH47, EXG1, PBS2 and a family of genes related to the human oxysterol binding protein, OSBP. CWH41 encodes a novel membrane N-glycoprotein located in the ER. Disruption of CWH41 leads to a K1 killer toxin resistant phenotype, and a 50% reduction in the $ beta$1,6-glucan level The $cwh{ it 41 /} Delta$ mutant displayed strong synergistic defects with $kre{ it 1 /} Delta$ or $kre{ it 1 /} Delta$ null mutations: the $cwh{ it 41 /} Delta kre{ it 6 /} Delta$ double mutant showed a slow-growth phenotype and a 75% reduction in $ beta$1,6-glucan level, and cells containing $cwh{ it 41 /} Delta kre{ it 6 /} Delta$ double mutations were nonviable. These results indicate that CWH41 is involved in the assembly of $ beta$1,6-glucan. / PTC1/CWH47 encodes a serine/threonine phosphatase, PBS2 is the structural gene for a MAPK kinase, and EXG1 codes for an exo-$ beta$-glucanase. Overexpression of EXG1 led to a killer resistant phenotype and a reduction in ($ beta$1,6-glucan level; whereas the $exg{ it 1 /} Delta$ mutant displayed modest increases in killer sensitivity and $ beta$1,6-glucan levels. Disruption of PTC1/CWH47 and overexpression of PBS2 resulted in similar $ beta$-glucan related phenotypes, with elevated EXG1 transcription, increased Exg1p activity, reduced $ beta$1,6-glucan levels, and resistance to killer toxin. The killer resistant phenotype caused by PTC1/CWH47 disruption or PBS2 overproduction were partially suppressed by the $exg{ it 1 /} Delta$ null mutation. These results suggest that Ptc1p/Cwh47p and Pbs2p play opposing regulatory roles in $ beta$-glucan assembly, and this is effected in part by modulating Exg1p activity. / Three yeast genes, KES1, HES1 and OSH1, whose products show homology to the human oxysterol binding protein, were also identified. Mutations in these genes resulted in sterol-related phenotypes, which include tryptophan-transport defects and nystatin resistance. In addition, mutant combinations showed small but cumulative reductions in membrane ergosterol levels. The three genes are also functionally related; since overexpression of HES1 or KES1 alleviated the tryptophan-transport defect in $kes{ it 1 /} Delta$ or $osh{ it 1 /} Delta$ mutants, respectively. These observations implicate the KES1-related gene family in ergosterol synthesis and provide comparative evidence of a role for human OSBP in cholesterol synthesis.

No description available.

Mutations in the yeast gene KRE1 lead to resistance to the K1 killer toxin of S. cerevisiae. The resistant phenotype is associated with a 40% reduction of the cell wall (1 $ to$ 6)-$ beta$-glucan fraction. Yeast cell wall (1 $ to$ 6)-$ beta$-glucan is a highly branched glucose polymer composed predominantly of linear (1 $ to$ 6)-$ beta$- sc D-linked glucopyranosyl residues. This glucan acts as a receptor for the killer toxin, leading to a concentration of active toxin on the yeast cell surface. The KRE1 gene was cloned by complementation of the kre1-1 mutant allele. The KRE1 gene encodes a serine and threonine rich protein, that is directed into the yeast secretory pathway, where it is highly modified, probably through O-linked glycosylation. Haploid strains with a kre1::HIS3 disruption appear to grow somewhat more slowly than wild type, and show an unusual wall structure when examined using electron microscopy. As with strains that carry a mutant kre1-1 allele those with a kre1::HIS3 disruption show a 40% reduced level of cell wall (1 $ to$ 6)-$ beta$-glucan. Structural comparison of the (1 $ to$ 6)-$ beta$-glucan fraction isolated from a wild type strain and a kre1 mutant, showed that the glucan obtained from the mutant had fewer (1 $ to$ 6)-linked residues and displayed a smaller average polymer size. Therefore, the KRE1 gene product appears to be required for the stepwise synthesis of cell wall (1 $ to$ 6)-$ beta$-glucan.

Compounds belonging to the vitamin K family possess anti-hemorrhagic property, and are used in treating patients suffering from hypoprothrombinemia and obstructive jaundice. Some of these compounds also exhibit marked antimicrobial activity toward various microorganisms. Vitamin K₅, 4-amino-2-methyl- 1-naphthol hydrochloride, a water-soluble analog of vitamin K has been shown to possess such an antimicrobial activity toward many bacteria, molds, and yeasts. Much of the work reported in literature is on its use as a possible food preservative, and little information is available on the mechanism of its action on the microorganisms. In this study, the mode of action of vitamin K₅ on Saccharomyces cerevisiae was investigated. Its effect on yeast cells, with and without sodium chloride; its color reactions with trisodium pentacyanoaminoferroate; its influence on certain enzyme systems of the yeast cell as indicated by 2, 3, 5-triphenyltetrazolium chloride; and the antagonistic effect of cysteine hydrochloride toward it were studied. In addition, the sulfhydryl groups of yeast cells were estimated by amperometric titration, and carbon dioxide production by yeast cells from different substrates was determined manometrically. The results showed that vitamin K₅ did not inhibit the yeast cells immediately on coming into contact with them. Vitamin K₅, with sodium chloride, produced 76 percent inhibition of yeast cells in 50 minutes as compared to 50 percent inhibition produced during the same time when used alone. Cysteine hydrochloride reacted with vitamin K₅ and stopped it from producing a blue color with trisodium pentacyanoaminoferroate. Cysteine hydrochloride also antagonized the antifungal activity of vitamin K₅, since in its presence vitamin K₅ failed to stop the reduction of 2, 3, 5-triphenyltetrazolium chloride by dehydrogenase systems. The yeast cells which had previously been inhibited by vitamin K₅ could not be revived by treatment with cysteine hydrochloride. These cells had developed a dark pink color which could not be removed by repeated washings. The sulfhydryl groups of yeast cells were reduced quantitatively when they were exposed to vitamin K₅. The reduction was not immediate and followed the same pattern as in case of total counts. Higher concentrations of vitamin K₅ inhibited carbon dioxide production by yeast cells to a greater extent in the three substrates used in this study. The action of vitamin K₅ is not immediate, the coloration picked up by the cells cannot be washed off, and its action is hastened in the presence of sodium chloride. All these seem to indicate that permeability of the cell is involved in some manner in the inhibition process. Higher concentrations of vitamin K₅ produce greater inhibition which shows that this compound also combines with sulfhydryl groups of the yeast cells. These findings suggest that the mode of action of vitamin K₅ is most likely due to an alteration of cell permeability as well as by combining with sulfhydryl groups of the yeast cells. / Graduation date: 1963

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Heidelberg, Univ., Diss., 2008.