The Saccharomyces cerevisiae ynl294c⌬ (rim21⌬) mutant was identified in our lab owing to its moderate resistance to calcofluor, although it also displayed all of the phenotypic traits associated with its function as the putative sensor (Rim21p) of the RIM101 pathway. rim21⌬ also showed moderate hypersensitivity to sodium dodecyl sulfate, caffeine, and zymolyase, and the cell wall compensatory response in this mutant was very poor, as indicated by the almost complete absence of Slt2 phosphorylation and the modest increase in chitin synthesis after calcofluor treatment. However, the cell integrity pathway appeared functional after caffeine treatment or thermal stress. rim21⌬ and rim101⌬ mutant strains shared all of the cell-wall-associated phenotypes, which were reverted by the expression of Rim101-531p, the constitutively active form of this transcription factor. Therefore, the absence of a functional RIM101 pathway leads to cell wall defects. rim21⌬, as well as rim101⌬, was synthetic lethal with slt2⌬, a synthetic defect alleviated by osmotic stabilization of the media. The double mutants grown in osmotically stabilized media were extremely hypersensitive to zymolyase and showed thicker cell walls, with poorly defined mannoprotein layers. In contrast, rim21⌬ rlm1⌬ and rim101⌬ rlm1⌬ double mutants were fully viable. Taken together, these results show that the RIM101 pathway participates directly in cell wall assembly and that it acts in parallel with the protein kinase C pathway (PKC) in this process independently of the transcriptional effect of the compensatory response mediated by this route. In addition, these results provide new experimental evidence of the direct involvement of the PKC signal transduction pathway through the Sltp2 kinase in the construction of yeast cell walls.

In Saccharomyces cerevisiae cytokinesis is efficiently achieved when a concerted series of events take place at the neck region, leading to septum formation. Here it is shown that Bni4p plays a crucial role in this process. Dbni4 mutants contain normal amounts of chitin and show normal chitin synthase III (CSIII) activity, but are partially resistant to Calcofluor White (CFW), probably due to the striking pattern of chitin distribution. CFW vital staining shows that chitin is synthesized in daughter cells and that it is also asymmetrically deposited at the mother-side of the neck in large-budded cells. This specific pattern coincides with that of Chs4p and Chs3p proteins. Alternatively, staining of unbudded cultures confirmed that Bni4p directs early chitin ring assembly, but is no longer required for the chitin deposition that occurs late in the cell cycle at cytokinesis. Consequently, this work provides a strategy to genetically discriminate between the absence of chitin synthesis (Dchs3 mutant) and failure in chitin ring assembly (Dbni4 mutants). The characterization of double mutants affected in chitin synthesis and primary septum (PS) assembly (Dmyo1 and Dchs2) provides evidence for the cooperation of Bni4p in PS formation besides its role in chitin ring assembly. In addition, it is shown that the chitin ring, but not the late deposition of chitin, cooperates in the correct assembly of the actomyosin ring and the PS when the biological function of the septins is compromised. We conclude that Bni4p is not only required for the assembly of the chitin ring, but is also involved in septum architecture and the maintenance of neck integrity.

We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-D-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-D-glucose-6-phosphate. Dough fermentation (CO 2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties.Saccharomyces cerevisiae may utilize a variety of carbon sources, but glucose and fructose are preferred. When one of these sugars is present, carbon catabolite repression occurs and the enzymes required for utilization of the alternative carbon sources are synthesized at low rates or not at all (12,13,14). Carbon catabolite repression alters transcription and is regulated mainly by the Mig1p protein (13, 14, 23), a transcriptional repressor of glucose-repressible genes involved in metabolic processes other than glucose fermentation (such as utilization of the alternative carbon sources sucrose, maltose, or galactose; gluconeogenesis; and respiratory metabolism [13,14]). Transcription of genes required for growth in nonfermentable carbon sources is activated by the Hap complex, which is repressed by Mig1p (5,14).S. cerevisiae baker's yeasts commonly are grown in molasses, which contains sucrose as the primary carbon source, and genotypes with the highest growth rate and productivity in molasses are favored (8,9,10,11). Further increases in invertase expression and redirection of the respiro-fermentative flux through the deregulation of Mig1p or Hap complex (5) would improve utilization of molasses and production of sweet doughs by these strains. Expression of the SUC genes, which code for the invertase required for catabolism of sucrose and raffinose, is repressed at high levels of glucose (12,13,14). Various regulatory regions have been identified in the SUC2 promoter. Mig1p binds to SUC2A and SUC2B (activation sequences) in the presence of glucose. Repression mediated by the upstream repression sequence for SUC2 (URS SUC2 ) occurs in the absence of glucose (14).In dough without addition of sugar, the principal fermentable sugar for yeast is maltose, liberated from the starch of the flour by amylases. The leavening ability of sponge dough is closely related to maltose fermentability (4,20). Maltose utilization requires a MAL locus and tr...

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