ENDOPLASMIC-RETICULUM MEMBRANE LOCALIZATION OF RCELP AND STE24P, YEAST PROTEASES INVOLVED IN CARBOXYL-TERMINAL CAAX PROTEIN PROCESSING AND AMINO-TERMINAL A-FACTOR CLEAVAGE

Proteins terminating in the CAAX motif, for example Ras and the yeast a-factor mating pheromone, are prenylated, trimmed of their last three amino acids, and carboxyl-methylated, The enzymes that mediate these activities, collectively referred to as CAAX processing components, ha ve been identified genetically in Saccharomyces cerevisiae. Whereas th e Ram1p/Ram2p prenyltransferase is a cytosolic soluble enzyme, sequenc e analysis predicts that the other CAAX processing components, the Rce 1p and Ste24p proteases and the Ste14p methyltransferase, contain mult iple membrane spans, To determine the intracellular site(s) at which C AAX processing occurs, we have examined the localization of the CAAX p roteases Rce1p and Ste24p by subcellular fractionation and indirect im munofluorescence. We find that both of these proteases are associated with the endoplasmic reticulum (ER) membrane. In addition to having a role in CAAX processing, the Ste24p protease catalyzes the first of tw o cleavage steps that remove the amino-terminal extension from the a-f actor precursor, suggesting that the first amino-terminal processing s tep of a-factor maturation also occurs at the ER membrane, The ER loca lization of Ste24p is consistent with the presence of a carboxyl-termi nal dilysine ER retrieval motif, although we find that mutation of thi s motif does not result in mislocalization of Ste24p, Because the ER i s not the ultimate destination for a-factor or most CAAX proteins, our results imply that a mechanism must exist for the intracellular routi ng of CAAX proteins from the ER membrane to other cellular sites.