Characterization of Gac1p, a regulatory subunit of protein phosphatase type I involved in glycogen accumulation in Sacch
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O R I GI N A L P A P E R
X. Wu á H. Hart á C. Cheng á P. J. Roach á K. Tatchell
Characterization of Gac1p, a regulatory subunit of protein phosphatase type I involved in glycogen accumulation in Saccharomyces cerevisiae
Received: 9 November 2000 / Accepted: 26 January 2001 / Published online: 14 March 2001 Ó Springer-Verlag 2001
Abstract 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 Gac1p are necessary and sucient 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 Gac1p are necessary for binding to Glc7p, while Asn356 and Tyr357 of Gac1p are necessary for binding to Gsy2p. These results suggest that Gac1p targets PP1 to its substrate Gsy2p and that Gac1p may alter the catalytic activity of PP1. Our data also show that overexpression of Gac1p aects glucose repression and ion homeostasis, two additional targets of GLC7, suggesting that multiple regulatory subunits compete for Glc7p binding in vivo.
Communicated by T. D. Fox X. Wu á H. Hart1 á K. Tatchell (&) Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130, USA E-mail: [email protected] Tel.: +1-318-6757769 Fax: +1-318-6755180 C. Cheng2 á P. J. Roach Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202-5122, USA Present addresses: Novartis Inc. Research Triangle Park, NC 27709, USA
1 2
Eli Lilly and Co., Indianapolis, IN 46285, USA
Keywords GLC7 á GAC1 á Yeast á Protein phosphatase á Glycogen synthase
Introduction In mammals, type 1 Ser/Thr protein phosphatase (PP1) regulates various cellular processes including glycogen metabolism (Cohen 1989), RNA splicing (Mermoud et al. 1994; Hirano et al. 1996), cell cycle control (Fernandez et al. 1992), and muscle contraction (Cohen 1989). The PP1 holoenzymes responsible for these diverse activities dier with respect to their regulatory or targeting subunits, which target the PP1 catalytic subunit to speci®c subcellular locations and/or alter its speci®c activity (Cohen and Cohen 1989; Hubbard and Cohen 1993). The glycogen-associated forms of PP1, which are heterodimers composed of the catalytic subunit PP1c and a glycogen-binding subunit, RGl/GM (Tang et al. 1991), GL (Doherty et al. 1995) or PTG (Printen et al. 1997), serve as the paradigm for this model. Thes
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