The mutant type I protein phosphatase encoded by glc7-1from Saccharomyces cerevisiaefails to interact productively with the GAC1 encoded regulatory subunit

Department of Microbiology, North Carolina State University, Raleigh 27695-7615.
Molecular and Cellular Biology (Impact Factor: 4.78). 03/1994; 14(2):896-905. DOI: 10.1128/MCB.14.2.896
Source: PubMed


Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen
synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation
of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant.
We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the
same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p
and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined
by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between
Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation
or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data
support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation
defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

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Available from: Kelly Tatchell, Aug 25, 2015
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    • "Yeast strains and media The yeast strains used in this study are listed in Supporting Information , Table S1 and are congenic to KT1112 [MATa leu2 ura3 his3 (Stuart et al. 1994)] and KT1113 (Frederick and Tatchell 1996). The ipl1-2 mutation (Chan and Botstein 1993) was introduced into the KT1112 background by seven serial backcrosses. "
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    ABSTRACT: Ipl1/Aurora B is the catalytic subunit of a protein kinase complex required for chromosome segregation and nuclear division. Before anaphase, Ipl1 is required to establish proper kinetochore-microtubule associations and to regulate the spindle assembly checkpoint (SAC). The phosphatase Glc7/PP1 opposes Ipl1 for these activities. To investigate Ipl1 and Glc7 regulation in more detail, we isolated and characterized mutations in the yeast Saccharomyces cerevisiae that raise the restrictive temperature of the ipl-2 mutant. These suppressors include three intragenic, second-site revertants in IPL1; 17 mutations in Glc7 phosphatase components (GLC7, SDS22, YPI1); two mutations in SHP1, encoding a regulator of the AAA ATPase Cdc48; and a mutation in TCO89, encoding a subunit of the TOR Complex 1. Two revertants contain missense mutations in microtubule binding components of the kinetochore. rev76 contains the missense mutation duo1-S115F, which alters an essential component of the DAM1/DASH complex. The mutant is cold sensitive and arrests in G2/M due to activation of the SAC. rev8 contains the missense mutation ndc80-K204E. K204 of Ndc80 corresponds to K166 of human Ndc80 and the human Ndc80 K166E variant was previously shown to be defective for microtubule binding in vitro. In a wild-type IPL1 background, ndc80-K204E cells grow slowly and the SAC is activated. The slow growth and cell cycle delay of ndc80-K204E cells are partially alleviated by the ipl1-2 mutation. These data provide biological confirmation of a biochemically based model for the effect of phosphorylation on Ndc80 function.
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    • "Reg1 controls glucose repression, growth and glycogen accumulation 28, 29, whereas Reg2 and Sds22 affect growth and cell-cycle progression 28, 30. Shp1, Gac1 and the Gac1-related proteins Gip2 and Pig1 appear to affect glycogen accumulation 31, 32, 33, 34. However, Gip1 is involved in meiosis and sporulation 35. "
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    ABSTRACT: The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.
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    • "Deletion of either PHO85 or both PCL8 and PCL10 results in hyperactivation of glycogen synthase and overaccumulation of glycogen (Huang et al. 1998; Timblin et al. 1996). Dephosphorylation and activation of Gsy2p is catalyzed by the Glc7p protein phosphatase in complex with the Gac1p targeting subunit (Feng et al. 1991; Francois et al. 1992; Stuart et al. 1994). "
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    ABSTRACT: The budding yeast, Saccharomyces cerevisiae, accumulates the storage polysaccharide glycogen in response to nutrient limitation. Glycogen synthase, the major form of which is encoded by the GSY2 gene, catalyzes the key regulated step in glycogen storage. Here, we utilized Gsy2p fusions to green fluorescent protein (GFP) to determine where glycogen synthase was located within cells. We demonstrated that the localization pattern of Gsy2-GFP depended upon the glycogen content of the cell. When glycogen was abundant, Gsy2-GFP was found uniformly throughout the cytoplasm, but under low glycogen conditions, Gsy2-GFP localized to discrete spots within cells. Gsy2p is known to bind to glycogen, and we propose that the subcellular distribution of Gsy2-GFP reflects the distribution of glycogen particles. In the absence of glycogen, Gsy2p translocates into the nucleus. We hypothesize that Gsy2p is normally retained in the cytoplasm through its interaction with glycogen particles. When glycogen levels are reduced, Gsy2p loses this anchor and can traffic into the nucleus.
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