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 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 subcellular localization of these proteins. A Rab3A mutant (T54A) that does not bind rabphifin 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.
McKiernan
C J
CJ
Department of Pathology, University of Vermont College of Medicine, Burlington 05405-0068, USA.
Stabila
P F
PF
Macara
I G
IG
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002118:Calcium / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D033921:Vesicular Transport Proteins; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D019204:GTP-Binding Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D015032:Zinc; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A