THE RAB3A GTPASE INTERACTS WITH MULTIPLE FACTORS THROUGH THE SAME EFFECTOR DOMAIN - MUTATIONAL ANALYSIS OF CROSS-LINKING OF RAB3A TO A PUTATIVE TARGET PROTEIN

Rab3A/smg25A is a small Ras-like guanine nucleotide binding protein im plicated in the control of regulated secretion from cells. Rab3A is ap proximately 30% cytosolic and 70% associated with the membranes of sec retory vesicles. It cross-links specifically to a rat brain membrane p rotein of about 85 kilodaltons (p85). To identify epitopes on Rab3A th at are important for its interaction with this putative target protein , we have determined the effects of point mutations on the cross-linki ng efficiency of Rab3A to p85. Rab3A, which was preincubated with a no n-hydrolyzable analog of GTP, cross-linked more efficiently to p85 tha n did Rab3A.GDP. Rab3A mutants that had decreased nucleotide binding a lso exhibited poor cross-linking to p85. Mutations in the effector dom ain, a site important for the interaction of Rab3A with its guanine nu cleotide releasing factor, guanine nucleotide dissociation inhibitor, and GTPase-activating protein, eliminated the ability of Rab3A to cros s-link to p85. However, short peptides corresponding to the effector d omain did not reduce cross-linking efficiency when present at a concen tration of 50 muM.