Cj. Mckiernan et al., ROLE OF THE RAB3A-BINDING DOMAIN IN TARGETING OF RABPHILIN-3A TO VESICLE MEMBRANES OF PC12 CELLS, Molecular and cellular biology, 16(9), 1996, pp. 4985-4995
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.