Abstract
The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1). Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays. Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested. Ypi1 contains a 48RHNVRW53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif. A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases. Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology. Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background. Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast.
Highlights
In eukaryotic organisms, protein phosphatases play a key role in the control and integration of cellular physiology
The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1)
Yfr003c Belongs to a Highly Conserved Family of Proteins Including a PP1 Protein Phosphatase Inhibitor—YFR003c encodes a small protein (155 residues; 18 kDa, estimated molecular mass) very rich in hydrophilic residues (Asp ϩ Glu content 19.4%; Ser ϩ Thr content 14.8%; Lys ϩ Arg content 16.8%) that shows an aberrant mobility in SDS-PAGE and that is heat-stable
Summary
Protein phosphatases play a key role in the control and integration of cellular physiology. More than 45 bona fide or putative PP1cregulating subunits have been defined in higher eukaryotes [15,16,17] These subunits are structurally quite different, but almost all of them present a consensus binding motif (R/K)(V/ I)X(F/W) necessary for PP1c regulation, which can account for the mutually exclusive binding of the different subunits to PP1c [15,16,17,18,19,20]. In a two-hybrid screening of a human brain cDNA library searching for potential mammalian PP1c regulatory proteins, a novel PP1 inhibitor, namely inhibitor-3, was identified. This protein shared 21% identity with a protein of unknown function encoded by the yeast YFR003c open reading frame [31]. This protein could be a good candidate for an endogenous inhibitor of Glc phosphatase activity
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