title: Protein carboxyl methylation in Saccharomyces cerevisiae: evidence for STE14-dependent and STE14-independent pathways.
abstract: We incubated yeast cells (Saccharomyces cerevisiae) with the methyl donor S-adenosyl-L-[methyl-3H]methionine and then fractionated their cellular components by gel electrophoresis in sodium dodecyl sulfate. By analyzing gel slices for [3H]methyl esters by a vapor-phase diffusion assay, we detect major methyl-esterified species that migrate at apparent polypeptide sizes of 24 and 22 kDa and minor species of 49, 38, 35, 33, 31, and 26 kDa. Incubation of extracts from labeled cells with ribonuclease A or proteinase K revealed that the 24- and 22-kDa species represent methyl-esterified RNAs, whereas the other species are methyl-esterified polypeptides. The 38-, 33-, 31-, and 26-kDa polypeptides were not methyl-esterified in an isogenic yeast strain lacking the STE14 gene encoding a C-terminal isoprenylcysteine methyltransferase, suggesting that they are substrates for the STE14 methyltransferase. On the other hand, the amount of the methylated 49-kDa polypeptide is reduced in the ste14 mutant, indicating that at least two methylated polypeptides are present--one a substrate of the STE14 methyltransferase and one a substrate of a STE14-independent methyltransferase. The 35-kDa polypeptide also appears to be methylated by a STE14-independent methyltransferase. When cells were incubated in the presence of the protein synthesis inhibitor cycloheximide, little or no methylation of the STE14-dependent species was detected while the methylation of the STE14-independent substrates was unaffected. Pulse-chase studies revealed significant turnover of all of the methylated species in a 4-h period, with the exception of the 38-kDa polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
authors.forename: C A
authors.initials: CA
authors.lastname: Hrycyna
authors.forename: M C
authors.initials: MC
authors.lastname: Yang
authors.forename: S
authors.initials: S
authors.lastname: Clarke
mesh_terms: D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters / Q000032:analysis; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D007553:Isotope Labeling; D008745:Methylation; D011496:Protein Methyltransferases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: D004952:Esters; D005656:Fungal Proteins; D003513:Cycloheximide; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase