In yeast, aging appears to be marked by a progressive impairment in cellular mechanisms, resulting in irreversible changes in physiology and morphology. To date, very little has been reported about the biochemical changes that occur in yeast as a function of individual cell aging. To investigate this further, six generations of a brewing yeast strain of Saccharomyces cerevisiae (NCYC 1239) were separated according to cellular age using continuous phased culturing and biotin-streptavidin magnetic cell sorting. / To obtain cells with no bud scars (virgin cells), a concentrated yeast slurry was layered onto sucrose density gradients and centrifuged. The uppermost band from the gradients was collected and cells were biotinylated with biotinamidocaproate- N-hydroxysuccinimide ester, that covalently binds to lysine residues on the yeast cell wall. For continuous phased culturing, biotinylated cells were added to a carbon-limited nutrient medium and growth was synchronized using the doubling time of the cells. Harvested cells were incubated with streptavidin superparamagnetic beads and sorted with a strong permanent magnet. In total, approximately 75% of the biotinylated cells were recovered. Viability testing was conducted using vital staining and plate counts, with >98% viability reported with the vital stain and 37% viability with the agar plates. / In conclusion, continuous phased culture, together with magnetic cell sorting has the potential to become a powerful tool for the study of age-related biochemical changes in yeast. Further studies will focus on ensuring the reproducibility of the method and using the recovered cells to study biochemical changes occurring during yeasts' replicative lifespan.

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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.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: In all cellular systems the ability to alter eellular programs in response to extracellular cues is essential for survival. This involves the integration of signals triggered by membrane bound receptors in order to adjust the expression of target genes and enzyme activities and consequently phenotypic outcome. The yeast Saccharomyces cerevisiae has evolved several adaptations, such as, sporulation and pseudohyphal differentiation, in order to survive changes in the surrounding environment. Pseudohyphal differentiation and the related phenotype, invasive growth, are proposed to be adaptations that enable the yeast to forage for scarce nutrients or escape from a detrimental environment. This dimorphic transition is associated with a change from the normal "yeast" form to a pseudohyphal form, which involves changes in budding pattern, cell-cycle progression, cellular elongation, and cell-eell and cell-substrate adherence. The outcome of these changes is elongated eells, which bud in a unipolar fashion and do not separate after budding to form chains of cells referred to as pseudohyphae. These pseudohyphae are able to penetrate the surface of agar containing growth medium, a process referred to as invasive growth. Nutrient-induced adaptations, such as pseudohyphal growth, have been extensively studied in S. cere visiae , and several factors have been implicated in the regulation thereof, many of which are part of specific signalling pathways. The most clearly defined are the filamentous growth specific MAP kinase cascade and the Gpa2p-cAMP-PKA pathway. MUC1/FL011, encoding a member of a family of cell wall associated proteins involved in cellcell/ cell-substrate adhesion, is regulated by these pathways and considered to be critical in the establishment of pseudohyphal differentiation and invasive growth. The promoter region of MUC1/FL011 represents one of the largest yeast promoters identified to date, with cis-acting elements present up to 2.4 kb upstream from the first coding triplet. The upstream regulatory region of MUC1/FL011 is almost identical to that of the STA2 gene, which encodes an extracellular glucoamylase required for the utilisation of extracellular starch. As suggested by the extent of homology between these two promoters, MUC1/FL011 and STA2 are co-regulated to a large degree and both require the same transcription factors. Mss11p plays a central role in the regulation of MUC1/FL011 and STA2 and consequently starch metabolism and pseudohyphaI differentiation. The regulation conferred by MSS11 on the transcriptional levels of MUC1/FL011 and STA2 also appears to be dependent on signals generated specifically in the presence of low nitrogen and glucose. Mss11p does not have significant homology to any other yeast protein, with the exception of limited homology to the transcriptional activator F108p. However, several distinctive domains have been identified in the MSS11 gene product. Firstly, Mss11p contains polyglutamine and poly-asparagine domains. It also contains a putative ATP- or GTP-binding domain (P-Ioop), commonly found in proteins such as kinases, ATPases or GTPases. Two short stretches close to the N-terminal, labelled H1 and H2, share significant homology to the transcriptional activator, F108p. Both the H2 domain and the extreme C-terminal of Mss11p are able to stimulate RNA polymerase II dependent transcription. Furthermore, the H1 domain together with the P-Ioop negatively regulates the activation potential of the H2 domain. This study presents further insight into the functioning of Mss11p and the involvement of the separate activation and regulatory domains in mediating transcriptional activation and pseudohyphal differentiation in response to nutrient limitation. Genetic interactions between Mss11p and other factors involved in the regulation of pseudohyphal growth and starch degradation were revealed, and specific regions of Mss11p were shown to be required by these factors in order to achieve their required function. In addition, results obtained in this study implicates Mss11p in the regulation of Ca2+-dependent flocculation and suggest that the FL01 gene is also regulated by Mss11p in this capacity. / AFRIKAANSE OPSOMMING: Die vermoë om sellulêre programme in reaksie op ekstrasellulêre seine te verander, is 'n essensiële vereiste vir alle sellulêre sisteme. Dit behels die integrasie van seine gegenereer deur membraan-gebonde reseptore om ekspressie van teikengene en ensiemaktiwiteite sodanig aan te pas dat gewenste fenotipise uitkomste bewerkstellig kan word. Die gis Saccharomyces cerevisiae het verskeie aanpassingsmeganismes ontwikkel, soos byvoorbeeld sporulasie en pseudohifeforming, om veranderinge in die omgewing te kan oorleef. Pseudohifevorming en die verwante fenotipe, penetrasiegroei, word beskou as aanpassings te wees wat die gis in staat stel om van 'n skadelike omgewing weg te kom, of dit in staat te stelom by skaars voedingstowwe uit te kom. Hierdie dimorfiese transisie word geassosieer met 'n verandering van die normale "gisvorm" tot pseudohifevorming wat veranderinge in die botpatroon, selsiklusprogressie, selverlenging, sel-sel en sel-substraat aanhegting behels. Die uitkoms van hierdie verandering is verlengde selle, wat unipolêr bot en nie van mekaar skei nie om sodoende kettings van selle te vorm en waarna verwys word as pseudohifes. Hierdie pseudohifes is ook in staat om die oppervlak van 'n agar bevattende groeimedium te penetreer, 'n proses waarna verwys word as penetrasiegroei. Aanpassings soos pseudohitevorminq is die afgelope dekade intensief nagevors, en verskeie faktore en seintransduksienetwerke is in die regulering daarvan geïmpliseer. Onder hierdie seintransduksienetwerke is die bes gedefiniëerde paaie die filamentasie-spesifieke MAP-kinasekaskade en die Gpa2p-cAMP-PKA pad. MUC1/FL011 kodeer vir 'n lid van 'n geenfamilie wat met sel-sel/sel-substraat aanhegting geasosieer word en dit word deur hierdie seintransduksie netwerke gereguleer. MUC1/FL011 word as essensieel vir pseudohife vorming beskou. MUC1/FL011 word gereguleer deur die grootste gispromoter wat tot op hede geïdentifiseer is, met cis-werkende elemente so ver as 2.4 kb stroom-op van ATG. Die MUC1/FL011 promoter is feitlik identies tot die van die STA2-geen, wat kodeer vir 'n ekstrasellulêre glukoamilase wat die gis in staat stelom ekstrasellulêre stysel te benut. Weens die homologie tussen die twee promoters, word MUC1/FL011 en STA2 tot In groot mate ge-koreguleer en beide benodig dieselfde transkripsiefaktore. Mss11p speel In sentrale rol in die regulering van MUC1/FL011 en STA2 en dus ook in die regulering van pseudohifevorming en styselmetabolisme. Die regulering wat deur Mss11p of MUC1/FL011 en STA2 uitgeofen word, blyk verder onderhewig te wees aan seine wat gegenereer word spesifiek in die teenwoordigheid van lae konsentrasies glukose en stikstof. Mss11p het nie betekenisvolle homologie met enige ander gisproteïen nie, behalwe vir beperkte homologie met die tranksripsionele aktiveerder F108p. Verskeie onderskeidbare domeine is egter in die MSS11 geenproduk teenwoordig. Eerstens, Mss11p bevat kenmerkende poliglutamien en poli-asparagien domeine. Verder bevat Mss11p ook In voorspelde ATP- of GTP-bindings domein (P-Ius), wat algemeen in proteïene soos kinases, ATPasaes en GTPases voorkom. Twee kort areas naby die N-terminaal, aangedui as H1 en H2, het betekenisvolle homologie met die transkripsiefaktor F108p. Beide die H2 domein en die ektreme C-terminaal van Mss11p is in staat om RNA polimerase " afhanklike transkripsie te stimuleer. Verder het die H1-domein in samewerking met die P-Ius In negatiewe uitwerking op die aktiveringspotensiaal van die H2-domein. Hierdie studie bied verdere insig tot die werking van Mss11p en die betrokkenheid van die verskeie aktiverings- en reguleringsdomeine by die oemiddetlinq van transkripsionele aktivering en pseudohifevorming in reaksie op beperking van voedingstowwe. Genetiese interaksies tussen Mss11p en ander faktore betrokke met die regulering van pseudohifevorming en styselafbraak is in hierdie studie aangetoon. Voorts is daar ook gewys dat spesifieke areas van Mss11p benodig word deur hierdie faktore om hulle biologiese funksie uit te oefen. Daar is ook In rol vir Mss11p in die regulering van Ca2+-afhanklike flokkulasie aangetoon en daar is bewys dat die FL01 geen deur Mss11p benodig word om hierdie effek uit te oefen.