The hypo-osmolarity-sensitive phenotype of the Saccharomyces cerevisiae hpo2 mutant is due to a mutation in PKC1, which regulates expression of beta-glucanase.
To obtain more information about the cell wall organization of Saccharomyces cerevisiae, we have developed a novel screening system to obtain cell wall-defective mutants, using a density gradient centrifugation method. Nine hypo-osmolarity-sensitive mutants were classified into two complementation groups, hpo1 and hpo2. Phase contrast microscopic observation showed that mutant cells bearing lesions at either locus became abnormally large. A gene that complemented the mutant phenotype of hpo2 was cloned and sequenced. This gene turned out to be identical to PKC1, which encodes the yeast homologue of mammalian protein kinase C. Complementation tests with pkc1 delta showed that hpo2 is allelic to pkc1. To study the reason for the fragility of hpo2 cells, cell wall was isolated and the glucan was analyzed. The amount of alkali, acid-insoluble glucan, which is responsible for the rigidity of the cell wall, was reduced to about 30% that of the wild-type cell and this may be the major cause of the fragility of the hpo2 mutant cell. Analysis of total wall proteins in hpo2 mutant cells on SDS-polyacrylamide gels revealed that a 33 kDa protein was overproduced two- to threefold relative to the wild-type level. This 33 kDa protein was identified as a beta-glucanase, encoded by BGL2. Disruption of BGL2 in the hpo2 mutant partially rescued the growth rate defect. This suggests that the PKC1 kinase cascade regulates BGL2 expression negatively and overproduction of the beta-glucanase is partially responsible for the growth defect.(ABSTRACT TRUNCATED AT 250 WORDS)
Shimizu
J
J
Central Research Laboratories, Asahi Breweries, Tokyo, Japan.
Yoda
K
K
Yamasaki
M
M
D002473:Cell Wall / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D043326:Glucan 1,3-beta-Glucosidase; D009154:Mutation; D010641:Phenotype; D011493:Protein Kinase C / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013696:Temperature; D014882:Water-Electrolyte Balance / Q000235:genetics; D001617:beta-Glucosidase / Q000235:genetics
D011493:Protein Kinase C; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase