Alanine-Scanning Mutagenesis of Protein Phosphatase Type 1 in the Yeast Saccharomyces cerevisiae

INVESTIGATIONS

Alanine-Scanning Mutagenesis of Protein Phosphatase Type 1 in the Yeast Saccharomyces cerevisiae

S. H. Baker, D. L. Frederick, A. Bloecher and K. Tatchell
Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695 Present address: Department of Biological Sciences, Clemson University, Clemson, SC 29634.

Protein phosphatase type 1, encoded by GLC7 in Saccharomyces cerevisiae, is an essential serine/threonine phosphatase implicated in the regulation of a diverse array of physiological functions. We constructed and examined 20 mutant alleles of GLC7 in which codons encoding clusters of charged residues were changed to alanine codons. Three of 20 mutant alleles alter residues in the active site of the phosphatase and are unable to rescue the lethality of a glc7::LEU2 disruption. The 17 alleles that support growth confer a range of mutant traits including cell cycle arrest, 2-deoxyglucose resistance, altered levels of glycogen, sensitivity to high salt, and sporulation defects. For some traits, such as 2-deoxyglucose resistance and cell cycle arrest, the mutated residues map to specific regions of the protein whereas the mutated residues in glycogen-deficient mutants and sporulation-defective mutants are more widely distributed over the protein surface. Many mutants have complex phenotypes, each displaying a diverse range of defects. The wide range of phenotypes identified from the collection of mutant alleles is consistent with the hypothesis that Glc7p-binding proteins, which are thought to regulate the specificity of Glc7p, have overlapping binding sites on the surface of Glc7p. This could account for the high level of sequence conservation found among type 1 protein phosphatases from different species.

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				N. T. Ramaswamy, L. Li, M. Khalil, and J. F. Cannon Regulation of Yeast Glycogen Metabolism and Sporulation by Glc7p Protein Phosphatase Genetics, May 1, 1998; 149(1): 57 - 72. [Abstract] [Full Text] [PDF]

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				H. Tachikawa, A. Bloecher, K. Tatchell, and A. M. Neiman A Gip1p-Glc7p phosphatase complex regulates septin organization and spore wall formation J. Cell Biol., November 26, 2001; 155(5): 797 - 808. [Abstract] [Full Text] [PDF]