Chitin synthesis is a process maintained across the fungal kingdom that, thanks to the power of genetic manipulation of yeast cells, is now beginning to be understood. Chitin synthesis is based on the regulation of distinct chitin synthase isoenzymes whose number ranges from one in Schizosaccharomyces pombe to seven in some filamentous fungi, such as Aspergillus fumigatus. This high diversity makes it difficult to find a unique model of regulation. However, the results available suggest common themes in regulation. The arrival of the genomic era, together with the development of fungal genetic technology should allow experimental approaches to this process.

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 ceUls caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescehce microscopy, in a S. cerevisiae wild-type cells during a-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglitinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in a-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 1-glucan in wild-type and m,utant strains were similar; however, the percentage of ,B-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 Saccharomyces cerevisiae CHS7 gene encodes an integral membrane protein located in the ER which is directly involved in chitin synthesis through the regulation of chitin synthase III (CSIII) activity. In the absence of CHS7 product, Chs3p, but not other secreted proteins, is retained in the ER, leading to a severe defect in CSIII activity and consequently, to a reduced rate of chitin synthesis. In addition, chs7 null mutants show the yeast phenotypes associated with a lack of chitin: reduced mating efficiency and lack of the chitosan ascospore layer, clear indications of Chs7p function throughout the S. cerevisiae biological cycle. CHS3 overexpression does not lead to increased levels of CSIII because the Chs3p excess is retained in the ER. However, joint overexpression of CHS3 and CHS7 increases the export of Chs3p from the ER and this is accompanied by a concomitant increase in CSIII activity, indicating that the amount of Chs7p is a limiting factor for CSIII activity. Accordingly, CHS7 transcription is increased when elevated amounts of chitin synthesis are detected.These results show that Chs7p forms part of a new mechanism specifically involved in Chs3p export from the ER and consequently, in the regulation of CSIII activity.