Department of Microbiology and Molecular Genetics
University of Pittsburgh School of Medicine
Pittsburgh, Pennsylvania 15261
| The phosphorylation status of Snf1 activation loop threonine is determined by changes in the rate of its dephosphorylation catalyzed by the yeast PP1 phosphatase Glc7 in a complex with the Reg1 protein. Previous studies have shown that Reg1 can associate with both Snf1 and Glc7 suggesting substrate binding as a mechanism for Reg1-mediated targeting of Glc7. In this study, the association of Reg1 with the three Snf1 isoforms was measured by two-hybrid analysis and coimmunoprecipitation. We found that Reg1 association with Snf1 occurred almost exclusively with the Gal83 isoform of the Snf1 complex. Nonetheless, Reg1 plays an important role in determining the phosphorylation status of all three Snf1 isoforms. We found that the rate of dephosphorylation for isoforms of Snf1 did not correlate with the amount of associated Reg1 protein. Functional chimeric beta subunits containing residues from Gal83 and Sip2 were used to map the residues needed to promote Reg1 association to the N-terminal 150 residues of Gal83. The Gal83 isoform of Snf1 is the only isoform capable of nuclear localization. A Gal83-Sip2 chimera containing the first 150 residues of Gal83 was able to associate with the Reg1 protein but did not localize to the nucleus. Therefore nuclear localization is not required for Reg1 association. Taken together, these data indicate that the ability of Reg1 to promote the dephosphorylation of Snf1 is not directly related to the strength of its association with the Snf1 complex. |
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Subcellular localization of beta subunits. A yeast strain lacking all three beta subunit genes was transformed with low-copy-number plasmids expressing either Gal83, Gal83-yEGFP, Sip2-yEGFP, or Gal83-Sip2-150-yEGFP, as indicated on the left. DIC and GFP fluorescence images were collected. Representative cells are shown. |