A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1

Abstract

Saccharomyces cerevisiae possesses a single essential gene (GLC7) encoding protein phosphatase 1 (PP1). Elevated expression of this gene from the GAL1 promoter is highly detrimental to the cell, causing a growth defect and aberrant bud morphology, which leads to cells exhibiting long, extended buds. By comparison, expression of GLC7 from the weaker MET3 promoter was without significant effect on either growth or morphology. However, repression of GLC7 expression from the MET3 promoter in cells where the MET3-GLC7 fusion was the sole source of PP1 resulted in a mitotic delay. Such cultures showed a massive decrease in the rate of proliferation in conjunction with a significant increase in the proportion of large, budded cells. 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and anti-tubulin immunofluorescence analysis of these cells revealed that many were blocked in mitosis, with a short spindle and DAPI-stained material stretched between the mother and daughter cell within the bud neck. These results support a role for PP1 in the completion of mitosis in S. cerevisiae.