Mutations in yeast protein phosphatase type 1 that affect targeting subunit binding

Protein phosphatase type 1 (PP1) is a major Ser/Thr protein phosphatase tha t is involved in many cellular processes. The activity of PP1 is controlled by regulatory subunits, many of which are thought to bind to a hydrophobic groove in PP1 via a short consensus sequence termed the V/IXF motif. To te st this hypothesis, 11 variants of yeast PP1 (Glc7) were constructed in whi ch one or more of the residues comprising the groove were changed to alanin e. These Variants were tested for their biological activity in vivo, for th eir biochemical activity in vitro, and for their ability to associate with three PP1 binding proteins. Five variants are unable to complement the esse ntial function of PP1 in vivo although they are catalytically active in vit ro. Many of the mutants are deficient in binding two V/IXF-containing subun its, Gac1 and Reg1, which regulate glycogen accumulation and glucose repres sion, respectively, but all retain the ability to associate with Sds22, a r egulatory subunit that lacks this motif. The subcellular locations at which PP1 normally accumulates (bud neck, nucleolus, spindle pole body) were not occupied by one PP1 variant. Additionally, we provide evidence that mutati ons in the hydrophobic groove of PPI affect substrate specificity. Together , these results demonstrate the importance of the hydrophobic groove for th e interaction with regulatory subunits, for the proper subcellular localiza tion of PP1 and for the substrate specificity of PP1.