PROTEIN CARBOXYL METHYLATION IN SACCHAROMYCES-CEREVISIAE - EVIDENCE FOR STE14-DEPENDENT AND STE14-INDEPENDENT PATHWAYS

We incubated yeast cells (Saccharomyces cerevisiae) with the methyl do nor S-adenosyl-L-[methyl-H-3] methionine and then fractionated their c ellular components by gel electrophoresis in sodium dodecyl sulfate. B y analyzing gel slices for [H-3]methyl esters by a vapor-phase diffusi on assay, we detect major methyl-esterified species that migrate at ap parent 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 wit h ribonuclease A or proteinase K revealed that the 24- and 22-kDa spec ies represent methyl-esterified RNAs, whereas the other species are me thyl-esterified polypeptides. The 38-, 33-, 31-, and 26-kDa polypeptid es 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. O n the other hand, the amount of the methylated 49-kDa polypeptide is r educed in the ste14 mutant, indicating that at least two methylated po lypeptides are present-one a substrate of the STE14 methyltransferase and one a substrate of a STE14-independent methyltransferase. The 35-k Da polypeptide also appears to be methylated by a STE14-independent me thyltransferase. When cells were incubated in the presence of the prot ein synthesis inhibitor cycloheximide, little or no methylation of the STE14-dependent species was detected while the methylation of the STE 14-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. These results demonstr ate the wide range of yeast substrates for the STE14 isoprenylcysteine methyltransferase and suggest the presence of at least one novel prot ein carboxyl methyltransferase in these cells.