The polybasic sequence in the C2B domain of rabphilin is required for the vesicle docking step in PC12 cells.
Rabphilin is generally thought to be involved in the regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, and it has recently been hypothesized that the C2B domain of rabphilin promotes the docking of dense-core vesicles to the plasma membrane through simultaneous interaction with a vesicle protein, Rab3A/27A, and a plasma membrane protein, SNAP-25 (synaptosome-associated protein of 25 kDa). However, the physiological significance of the rabphilin-SNAP-25 interaction in the vesicle-docking step has never been elucidated. In this study we demonstrated by a mutation analysis that the polybasic sequence (587 KKAKHKTQIKKK 598) in the C2B domain of rabphilin is required for SNAP-25 binding, and that the Asp residues in the Ca(2+)-binding loop 3 (D628 and D630) of the C2B domain are not required. We also investigated the effect of Lys-->Gln (KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in individual PC12 cells. A rabphilin(KQ) mutant that completely lacks SNAP-25-binding activity significantly decreased the number of plasma-membrane-docked vesicles and strongly inhibited high-KCl-induced dense-core vesicle exocytosis. These results indicate that the polybasic sequence in the C2B domain functions as an effector domain for SNAP-25 and controls the number of 'releasable' vesicles docked to the plasma membrane.
Tsuboi
Takashi
T
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan.
Kanno
Eiko
E
Fukuda
Mitsunori
M
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005089:Exocytosis / Q000502:physiology*; D008561:Membrane Fusion / Q000502:physiology; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009489:Neurosecretion / Q000502:physiology*; D016716:PC12 Cells; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D009419:Nerve Tissue Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D000097624:Rabphilin-3A; C494210:Snap25 protein, rat