The C2B domain of rabphilin directly interacts with SNAP-25 and regulates the docking step of dense core vesicle exocytosis in PC12 cells.
Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) J. Biol. Chem. 279, 13065-13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (i.e. Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca(2+) (EC(50) = 0.817 microm SNAP-25), but 0.5 mm Ca(2+) increases the affinity for SNAP-25 2-fold (EC(50) = 0.405 microm SNAP-25) without changing the B(max) value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-DeltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.
Tsuboi
Takashi
T
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Fukuda
Mitsunori
M
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005089:Exocytosis / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; D019204:GTP-Binding Proteins; C512962:Rab27a protein, mouse; C512961:Rab27a protein, rat; D020691:rab GTP-Binding Proteins