THE PUTATIVE SWITCH-2 DOMAIN OF THE RAS-RELATED GTPASE, RAB1B, PLAYS AN ESSENTIAL ROLE IN THE INTERACTION WITH RAB ESCORT PROTEIN

Posttranslational modification of Rab proteins by geranylgeranyltransf erase type II requires that they first bind to Rab escort protein (REP ). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Ra b proteins that are in the GDP state, but the specific structural doma ins involved in this interaction have not been defined. In p21 Ras, th e alpha 2 helix of the Switch 2 domain undergoes a major conformationa l change upon GTP hydrolysis. Therefore, we hypothesized that the corr esponding region in Rab1B might play a key role in the interaction wit h REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative alpha 2 helix of Myc-tagged Rab1B prevented prenyla tion of the recombinant protein in cell-free assays, whereas mutations in the alpha 3 and alpha 4 helices did not. Additionally, upon transi ent expression in transfected HEK-293 cells, the Myc-Rab1B alpha 2 hel ix mutants were not efficiently prenylated as determined by incorporat ion of [H-3]mevalonate. Metabolic labeling studies using [P-32]orthoph osphate indicated that the poor prenylation of the Rab1B alpha 2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtrat ion analysis of cytosolic fractions from 293 cells that were coexpress ing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed t hat mutations in the alpha 2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate t hat the Switch 2 domain of Rab1B is a key structural determinant for R EP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.