Protein phosphatase methyltransferase 1 (Ppm1p) is the sole activity responsible for modification of the major forms of protein phosphatase 2A in yeast.
Protein phosphatase 2A (PP2A) is a major threonine/serine phosphatase that is involved in regulating a variety of cellular processes. It has been shown in both yeast and mammals that the PP2A catalytic subunit (PP2Ac) is methyl-esterified at the conserved C-terminal Leu residue. The recent characterization of a mammalian PP2A carboxyl methyltransferase has led to the identification of two ORFs in Saccharomyces cerevisiae as potential orthologues of the mammalian PP2A methyltransferase: protein phosphatase methyltransferase 1 (PPM1) and protein phosphatase methyltransferase 2 (PPM2). To experimentally identify the PP2A methyltransferase in yeast, we obtained deletion mutants of PPM1 and PPM2 and then constructed double mutants. Using in vivo-labeling techniques, we demonstrate that only the PPM1 gene is required for PP2Ac methylation at the C-terminus. Because yeast has at least three homologues of PP2Ac (PPH21, PPH22, and PPH3), we then asked whether all of these catalytic subunits are methylated by the PPM1 and/or PPM2 putative methyltransferases. We modified the segment corresponding to the N-terminal coding region of all three PP2Ac genomic genes with a hemagglutinin (HA) tag in the parent, ppm1, ppm2, and ppm1ppm2 mutant genetic backgrounds. Using immuoprecipitation with anti-HA antibodies followed by methyl ester analysis, we showed that only in the ppm1 mutant were both Pph21p and Pph22p not methylated. We did not detect any methylesterification of Pph3p under our conditions. Our results indicate that PPM1 is the sole methyltransferase responsible for methylating the two major homologues of PP2Ac in yeast. The function of the PPM2 gene product remains unclear.
Kalhor
H R
HR
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Luk
K
K
Ramos
A
A
Zobel-Thropp
P
P
Clarke
S
S
D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D017353:Gene Deletion; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011496:Protein Methyltransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
D000939:Epitopes; D029701:Saccharomyces cerevisiae Proteins; C442645:Ppm1 protein, S cerevisiae; D011496:Protein Methyltransferases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2