Characterization of Gac1p, a regulatory subunit of protein phosphatase type I involved in glycogen accumulation in Saccharomyces cerevisiae.
GAC1 and GLC7 encode regulatory and catalytic subunits, respectively, of a type 1 phosphatase (PP1) in Saccharomyces cerevisiae that controls glycogen synthesis by regulating the phosphorylation state of glycogen synthase (Gsy2p). To investigate the role of Gac1p in this process, a set of GAC1 deletions were tested for their ability to complement a gac1 null mutation and to associate with Glc7p and with Gsy2p. The N-terminal 93 amino acids of Gaclp are necessary and sufficient for the interaction with Glc7p, whereas a region spanning residues 130-502 is required for Gsy2p binding. Both domains are required for full activity in vivo, although the Glc7p-binding domain retains some residual activity and can alter the phosphorylase a phosphatase activity of Glc7p in vitro. Further mutational analysis showed that Val71 and Phe73 of Gaclp are necessary for binding to Glc7p, while Asn356 and Tyr357 of Gaclp are necessary for binding to Gsy2p. These results suggest that Gac1p targets PPI to its substrate Gsy2p and that Gac1p may alter the catalytic activity of PP . Our data also show that overexpression of Gac1p affects glucose repression and ion homeostasis, two additional targets of GLC7, suggesting that multiple regulatory subunits compete for Glc7p binding in vivo.
Wu
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130, USA.
Hart
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Cheng
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Roach
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Tatchell
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D019943:Amino Acid Substitution; D002384:Catalysis; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006003:Glycogen / Q000378:metabolism*; D006006:Glycogen Synthase / Q000378:metabolism; D006706:Homeostasis; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; C073060:GAC1 protein, S cerevisiae; D010446:Peptide Fragments; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D006003:Glycogen; D006006:Glycogen Synthase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1