THE SACCHAROMYCES-CEREVISIAE PRENYLCYSTEINE CARBOXYL METHYLTRANSFERASE STE14P IS IN THE ENDOPLASMIC-RETICULUM MEMBRANE

Eukaryotic proteins containing a C-terminal CAAX motif undergo a serie s of posttranslational CAAX-processing events that include isoprenylat ion, C-terminal proteolytic cleavage, and carboxyl methylation. We dem onstrated previously that the STE14 gene product of Saccharomyces cere visiae mediates the carboxyl methylation step of CAAX processing in ye ast. Ln this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal a ntibody generated against the C terminus of Ste14p and an in vitro met hyltransferase assay. We demonstrate by immunofluorescence and subcell ular fractionation that Ste14p and its associated activity are localiz ed to the endoplasmic reticulum (ER) membrane of yeast. Ln addition, o ther studies from our laboratory have shown that the CAAX proteases ar e also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, pres umably on its cytosolic face. Interestingly, the insertion of a hemagg lutinin epitope tag at the N terminus, at the C terminus, or at an int ernal site disrupts the ER localization of Ste14p and results in its m islocalization, apparently to the Golgi. We have also expressed the St e14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Delta ste14 mutant. This find ing, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of function al homologues.