Expression of human protein phosphatase-1 in Saccharomyces cerevisiae highlights the role of phosphatase isoforms in regulating eukaryotic functions.
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.
Gibbons
Jennifer A
JA
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
Kozubowski
Lukasz
L
Tatchell
Kelly
K
Shenolikar
Shirish
S
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic; D006003:Glycogen / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D054645:Protein Phosphatase 1; D021122:Protein Subunits / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D013379:Substrate Specificity
D007527:Isoenzymes; D021122:Protein Subunits; D006003:Glycogen; D004247:DNA; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1