Expression of Human Protein Phosphatase-1 in Saccharomyces cerevisiae Highlights the Role of Phosphatase Isoforms in Regulating Eukaryotic Functions

Jennifer A Gibbons,Kelly Tatchell,Shirish Shenolikar,Lukasz Kozubowski

doi:10.1074/jbc.m701272200

Abstract

Human (PP1) isoforms, PP1alpha, PP1beta, PP1gamma1, and PP1gamma2, differ in primary sequences at N and C termini that potentially bind cellular regulators and define their physiological functions. The GLC7 gene encodes the PP1 catalytic subunit with >80% sequence identity to human PP1 and is essential for viability of Saccharomyces cerevisiae. In yeast, Glc7p regulates glycogen and protein synthesis, actin cytoskeleton, gene expression, and cell division. We substituted human PP1 for Glc7p in yeast to investigate the ability of individual isoforms to catalyze Glc7p functions. S. cerevisiae expressing human PP1 isoforms were viable. PP1alpha-expressing yeast grew more rapidly than strains expressing other isoforms. On the other hand, PP1alpha-expressing yeast accumulated less glycogen than PP1beta-or PP1gamma1-expressing yeast. Yeast expressing human PP1 were indistinguishable from WT yeast in glucose derepression. However, unlike WT yeast, strains expressing human PP1 failed to sporulate. Analysis of chimeric PP1alpha/beta subunits highlighted a critical role for their unique N termini in defining PP1alpha and PP1beta functions in yeast. Biochemical studies established that the differing association of PP1 isoforms with the yeast glycogen-targeting subunit, Gac1p, accounted for their differences in glycogen synthesis. In contrast to human PP1 expressed in Escherichia coli, enzymes expressed in yeast displayed in vitro biochemical properties closely resembling PP1 from mammalian tissues. Thus, PP1 expression in yeast should facilitate future structure-function studies of this protein serine/threonine phosphatase.

Highlights

(1), glycogen metabolism [2], and protein translation [3]
Saccharomyces cerevisiae is unique in that it is the only known eukaryote with a single PP1 gene, GLC7, which is essential for viability [9]
Human PP1 expressed in S. cerevisiae demonstrated biochemical properties more closely resembling PP1 from mammalian tissues than the enzyme expressed in E. coli, and provided a novel expression system that should greatly facilitate future mechanistic studies of this protein serine/threonine phosphatase

Summary

EXPERIMENTAL PROCEDURES

Materials—DNA was purified using QIAquick extraction, PCR purification kits, and QIAprep spin miniprep kits (Qiagen). PP1 genotypes were confirmed by colony PCR and expression of Glc7p and human PP1 confirmed by immunoblotting yeast lysates. JG40-49, were generated by integrating the PP1-expressing plasmid into the HIS3 locus of KT1900 and selecting strains for loss of the GFP-Glc7p-expressing maintenance plasmid by growth on CSM containing 5-FOA. The CUP1 promoter was used to express Glc7p and human PP1 catalytic subunits in yeast with the exception of plasmids derived from pJG24-29, which used the native GLC7 promoter and terminator (derived from p1855). PP1 catalytic subunits were partially purified from yeast strains. Assays with PP1 catalytic subunits expressed in E. coli included 1 mM MnCl2. PP1 Binding by Regulators—Sedimentation of human PP1 isoforms from yeast lysates using GST fusions of targeting subunits was undertaken as described [26]. Immunoreactivity was quantified using anti-mouse AlexaFluor 680 (Molecular Probes) and anti-rabbit IR Dye 800 (Rockland) secondary antibodies, respectively, using Odyssey infrared imaging system (Li-COR Biosciences)

RESULTS

Biochemical Analysis of Human

Although partially purified human

DISCUSSION