Role of the Rab3A-Binding Domain in Targeting of Rabphilin-3A to Vesicle Membranes of PC12 Cells

Abstract

Rab3A is a small GTPase implicated in the docking of secretory vesicles in neuroendocrine cells. A putative downstream target for Rab3A, rabphilin-3A, is located exclusively on secretory vesicle membranes. It contains near its C terminus two C₂ domains that bind Ca²⁺ in a phospholipid-dependent manner and an N-terminal, Rab3A-binding domain that includes 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 consists of residues 45 to 170 of rabphilin. HA1-tagged Rab3A and a green fluorescent protein (GFP)-rabphilin fusion were used to examine the roles of Rab3A and of rabphilin domains in the sub-cellular 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 C₂ domains of rabphilin reduced membrane association of GFP-rabphilin but did not cause mistargeting of the membrane-associated fraction. However, disruption of the zinc fingers, which drastically reduced Rab3A binding, did not reduce membrane association. These results suggest that the C₂ domains are required for efficient membrane attachment of rabphilin in PC12 cells and that Rab3A binding may act to target the protein to the correct membrane.