The regulation of glycogen metabolism and ion transport by protein phosphatase type 1 in Saccharomyces cerevisiae.

摘要

Protein phosphatase type 1 (PP1) is encoded by the essential gene GLC7 in Saccharomyces cerevisiae. The glc7-109 (K259A, R260A) mutant has a dominant, hyperglycogen defect and recessive ion and drug sensitivities, which reflect that Glc7 regulates glycogen metabolism and ion transport in yeast. The R260 residue in GLC7 is responsible for the dominant glycogen and recessive salt traits of glc7-109 and these defects are due to defects in distinct physiological pathways. The hyperglycogen phenotype of this mutant is partially retained in null mutants of GAC1, the glycogen-specific targeting subunit of GLC7. However, deletion of GAC1 homologues, GIP2 and/or PIG2 suppresses the glycogen defects of the glc7-109 mutant and in the yeast two-hybrid assay, the Glc7-109 protein interacts with Gac1, Gip2 and Pig2. Moreover, cells containing the R73C mutation (glc7-1) accumulate very low glycogen levels and Glc7-1 does not interact with Gac1, Gip2, or Pig2. Furthermore, the glc7-F256A mutant accumulates only slightly lower than wild-type glycogen levels and Glc7-F256A fails to bind to Gac1, but interacts with Gip2 and Pig2. Together, these data suggest that GIP2 and PIG2 may encode Glc7-regulatory subunits that regulate glycogen metabolism.; The glc7-109 mutant is sensitive to canons, aminoglycosides, and alkaline pH and exhibits increased rate of uptake of 3,3′ -dihexyloxacarbocyanine iodide uptake, a fluorescent dye that indicates relative membrane potential. The glc7-109 is also resistant to molar concentrations of sorbitol or KCl, indicating that it has normal osmoregulation. KCl, CaCl₂ and mutations in the plasma membrane H+ATPase, Pma1, suppress the cation and drug sensitivities of the glc7-109 mutant. Genetic and biochemical characterization of the glc7-109 mutant indicates that it has a hyperpolarized membrane and its defects are independent of calcineurin, a phosphatase that transcriptionally regulates ion transporters; Pma1, which regulates intracellular pH and the membrane potential; and K+ transporters, Trk1 and Trk1, which regulate K+ uptake and membrane potential. The glc7-109 and glc7-F256A mutants have different cation and drug sensitivities, which may reflect differences in the nature of their ion transport defects. Suppressor analysis of both mutants implicates the Yck1 and Yck2 yeast casein kinase 1 isoforms, a putative drug: H + antiporter and a transcriptional repressor, Mig1 in the regulation of Glc7-dependent ion transport.