Ksg1, a homologue of the phosphoinositide-dependent protein kinase 1, controls cell wall integrity in Schizosaccharomyces pombe

It has previously been shown that the Schizosaccharomyces pombe mutant ksg1-358 has a mating and sporulation defect at 30 degrees C and that it is temperature sensitive for growth at 35 degrees C. However the molecular basis for these phenotypes remained largely unknown. In this study we show that ksg1-358 mutant cells lysed at the non-permissive temperature, which could be prevented by sorbitol. Overexpression of ksg1 using the nmt1-promoter showed slow growth and cells became swollen when incubated at 35 degrees C under low inositol conditions. Interestingly, in a two-hybrid assay we found that the ksg1-protein interacted with Pck1p, a protein implicated in regulating cell wall integrity in S. pombe. Genetic complementation assays showed that an overexpression of pck2, the homologue of pck1 involved in the regulation of cell wall synthesis, could partially rescue ksg1-358 phenotypes. We digested the ksg1-358 cell wall using beta-glucanase. We found that the ksg1-358 mutant was more resistant to cell lysis at 30 degrees C than the wildtype strain h972, which was similar to a pck1-deletion strain. A ksg1-overexpressing strain was hypersensitive towards beta-glucanase treatment similar to a pck2-deletion strain. The pck1-deletion partially rescued beta-glucanase hypersensitivity of the ksg1-overexpressing strain but the pck2-deletion increased it. The ksg1-358 mutation increased beta-glucanase resistance of a pck1-overexpressing strain but it had no effect on a pck2-overexpressing strain. Our results provide evidence that ksg1 is a novel regulator of cell wall integrity in the fission yeast Schizosaccharomyces pombe. They further suggest that Ksg1p acts in a pathway with Pck1p, possibly upstream and through direct interaction.