CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae.

Abstract

Abstract. The CALI 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 calRI 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 syn-HE antifungal effect of Calcofluor White (33, 34), a substance that binds specifically to chitin in vivo (10), was used to isolate and characterize several Saccharo myces cerevisiae mutants resistant to this fluorochrome (35) . The most interesting feature of these mutants was a defect in chitin synthesis in vivo not only during the vegetative cycle but also in response to the pheromone a-factor (35) . A defect in spore maturation in the homozygous condition was observed (35). The possibility that these mutants could define physiologically relevant genes involved in chitin synthesis was apparent; therefore, the cloning and sequencing of the gene corresponding to one of them, cal"l, was undertaken . In this report we show that the protein sequence encoded by the cloned gene (CAI/) has significant similarities with those of chitin synthase 1 (9) and chitin synthase 2 (42, 43) . Recently, Bulawa and Osmond (8) detected the presence of a third chitin synthase (chitin synthase 3 or Chs31) in strains defective in chitin synthase 1 and 2. We report here that Reprint requests should be addressed to Enrico Cabib, National Institutes of Health, Building 10, Room 9N-115, Bethesda, MD 20892 .A preliminary report of parts of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology held at The Hague, Netherlands, 1990.1. Abbreviations used in this paper: Chsl-3, chitin synthase 1-3 ; canChsl, chitin synthase 1 from Candida albicans .© The Rockefeller University Press, 0021-9525/91/07/101/9 $2 .00 The Journal of Cell Biology, Volume 114, Number 1, July 1991101-109 thase 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 CALI 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 calRI 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 CALI gene was not lethal, indicating that chitin synthase 3 is not an essential enzyme for S. cerevisiae. ca1R1 mutants are deficient in chiti...

Molecular analysis of Chs3p participation in chitin synthase III activity

“…S. cerevisiae strain W303 (MATa trp1 leu2 ura3 his3 ade2) was used as wild-type control. Strain TCY1 (MATa trp1 leu2 ura3 his3 ade2 chs3::LEU2) was created by LEU2 replacement of CHS3 as follows: the CHS3 gene from pHV7 [11] was cloned as an EcoRIϪ SphI fragment of 5.6 kb into the Stratagene vector pBluescript II KS (ϩ) (plasmid pTC1). The XhoIϪPstI fragment from CHS3 (nucleotides 519Ϫ2909) from pTC1 [12] was replaced by the LEU2 marker; this plasmid was digested with MluI and the 4.6-kb fragment that included the chs3::LEU2 construct was purified and transformed into strain W303 [13].…”

Sporulation in the Budding Yeast Saccharomyces cerevisiae

“…Similar to the b-1,3 glucan chains of the b-glucan layer, the b-1,4-linked glucosamine chains of the chitosan layer are synthesized by an integral membrane enzyme that binds nucleotide sugars in the cytoplasm and extrudes the polymer to the extracellular space (Orlean 1997). In vegetative cells, cell wall chitin is produced by three different chitin synthases: Chs1, Chs2, and Chs3 (Silverman et al 1988;Cabib et al 1989;Silverman 1989;Shaw et al 1991;Valdivieso et al 1991). During sporulation, chitosan synthesis is mediated solely by Chs3 (Pammer et al 1992).…”

Characterization of the essential yeast gene encoding N‐acetylglucosamine‐phosphate mutase

“…Kronstad et al (1987) demonstrated that at least two copies of the pheromone-responsive element were required for the full level a-factorenhanced BAR1 gene expression. Interestingly, pheromoneresponsive elements could also be identified in the upstream regions of three genes involved in chitin synthesis: GFAU GCNl encoding L-glutamine:fructose-6-phosphate amidotransferase (Watzele and Tanner, 1989), CHSl encoding chitin-synthase I (Bulawa et al, 1986;Appeltauer and Achsstetter, 1989) and CSD2 which is essential for chitin synthase 111 activity (Valdivieso et al, 1991 ;Orlean, 1987). Furthermore, Orlean et al (1985) observed that mating factors increase the de novo synthesis of amino sugars.…”