Post-translational processing and membrane association of the two early endosome-associated rab GTP-binding proteins...

The two early endosome-associated rab GTP-binding proteins, rab4 and rab5, are suggested to regulate endocytosis. In this report, we examined post-translational processing and membrane association of the two rab proteins. Human rab4 and rab5 were expressed in chicken embryo fibroblasts using a Sindbis virus expression vector. Cells were labeled with either [35S]methionine or [3H]mevalonolactone. Cell lysates were immunoprecipitated with antisera specific for rab4 and rab5, respectively, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that both rab4 and rab5 contained at least three forms: the precursor form, the isoprenylated intermediate, and the isoprenylated mature form of faster mobility. The rab5 intermediate comigrated with the precursor form, whereas the rab4 intermediate migrated slightly faster than the precursor form. The intermediate form of rab4, but not rab5, accumulated in the presence of a chymotrypsin-like protease inhibitor (N-acetyl Tyr ethyl ester), suggesting that proteolysis was required for generation of the mature form. Furthermore, the intermediate and mature forms of rab4, but not rab5, were carboxyl-methylated as demonstrated by incorporation of alkali-labile counts from [methyl-3H]methionine. Membrane association of the distinct rab4 and rab5 forms was examined by subcellular fractionation and Triton X-114 partitioning. The precursor forms were found in the cytosol and partitioned into the aqueous phase. The mature forms were membrane-associated and partitioned into the detergent phase. Unexpectedly, the isoprenylated intermediate forms of both rab4 and rab5 partitioned exclusively into the aqueous phase. Taken together, the data indicate that the entire post-translational processing, which includes isoprenylation, carboxyl methylation (rab4), and possibly proteolysis, confers the competency for membrane association of rab4 and rab5.