The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.

Cyclins regulate the major cell cycle transitions in eukaryotes through association with cyclin-dependent protein kinases (CDKs). In yeast, G₁ cyclins are essential, rate-limiting activators of cell cycle initiation. G₁-specific accumulation of one G₁ cyclin, Cln2, results from periodic gene expression coupled with rapid protein turnover. Site-directed mutagenesis of CLN2 revealed that its phosphorylation provides a signal that promotes rapid degradation. Cln2 phosphorylation is dependent on the Cdc28 protein kinase, the CDK that it activates. These findings suggest that Cln2 is rendered self-limiting by virtue of its ability to activate its cognate CDK subunit.

The CAL1 gene was cloned by complementation of the defect in Calcofluor-resistant calR1 mutants of Saccharomyces cerevisiae. Transformation of the mutants with a plasmid carrying the appropriate insert restored Calcofluor sensitivity, wild-type chitin levels and normal spore maturation. Southern blots using the DNA fragment as a probe showed hybridization to a single locus. Allelic tests indicated that the cloned gene corresponded to the calR1 locus. The DNA insert contains a single open-reading frame encoding a protein of 1,099 amino acids with a molecular mass of 124 kD. The predicted amino acid sequence shows several regions of homology with those of chitin synthases 1 and 2 from S. cerevisiae and chitin synthase 1 from Candida albicans. calR1 mutants have been found to be defective in chitin synthase 3, a trypsin-independent synthase. Transformation of the mutants with a plasmid carrying CAL1 restored chitin synthase 3 activity; however, overexpression of the enzyme was not achieved even with a high copy number plasmid. Since Calcofluor-resistance mutations different from calR1 also result in reduced levels of chitin synthase 3, it is postulated that the products of some of these CAL genes may be limiting for expression of the enzymatic activity. Disruption of the CAL1 gene was not lethal, indicating that chitin synthase 3 is not an essential enzyme for S. cerevisiae.

Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter cells caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescence microscopy, in a S. cerevisiae wild-type cells during alpha-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglutinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in alpha-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and beta-glucan in wild-type and mutant strains were similar; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to a cell wall enzymic lytic complex were rather similar. The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.