ROLE OF THE RAB3A-BINDING DOMAIN IN TARGETING OF RABPHILIN-3A TO VESICLE MEMBRANES OF PC12 CELLS

Rab3A is a small GTPase implicated in the docking of secretory vesicle s in neuroendocrine cells. A putative downstream target for Rab3A, rab philin-3A, is located exclusively on secretory vesicle membranes. It c ontains near its C terminus two C-2 domains that bind Ca2+ in a phosph olipid-dependent manner and an N-terminal, Rab3A-binding domain that i ncludes a Cys-rich region. We have determined that the Cys-rich domain binds two Zn2+ ions and is necessary but not sufficient for efficient binding of rabphilin to Rab3A. A minimal Rab3A-binding domain consist s of residues 45 to 170 of rabphilin. HA1-tagged Rab3A and a green flu orescent protein (GFP)-rabphilin fusion were used to examine the roles of Rab3A and of rabphilin domains in the subcellular localization of these proteins. A Rab3A mutant (T54A) that does not bind rabphilin in vitro colocalized with the GFP-rabphilin fusion, indicating that Rab3A targeting is independent of its interaction with rabphilin. Deletion of the C-2 domains of rabphilin reduced membrane association of GFP-ra bphilin but did not cause mistargeting of the membrane-associated frac tion. However, disruption of the zinc fingers, which drastically reduc ed Rab3A binding, did not reduce membrane association. These results s uggest that the C-2 domains are required for efficient membrane attach ment of rabphilin in PC12 cells and that Rab3A binding may act to targ et the protein to the correct membrane.