Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.

Abstract

GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage‐dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP‐rab3 via a N‐terminal domain comprising a novel Zn2+(‐)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage‐dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction.

Rabphilin Potentiates SolubleN-Ethylmaleimide Sensitive Factor Attachment Protein Receptor Function Independently of rab3

“…Previous studies in vertebrates have found that vertebrate rabphilin is recruited to vesicles through its association with rab3 (Stahl et al, 1996), whereas other studies have found localization of rabphilin to be independent of an interaction with rab3 . We examined whether RBF-1 localization is mediated through rab3.…”

“…1C). The deleted region includes the start codon and the first two CXXC motifs that have been implicated previously in the binding of Zn 2ϩ and rab3 (McKiernan et al, 1996;Stahl et al, 1996). Although a transcript derived from the mutant locus can be detected using reverse transcriptase-PCR, the proximal ATG of this transcript is out of frame with the remaining RBF-1 coding sequences.…”

“…Models for rabphilin function have postulated that rab3 recruits rabphilin to synaptic vesicles (Li et al, 1994;Stahl et al, 1996;Ostermeier and Brunger, 1999). However, in C. elegans, we have shown that this is not the case: rab-3 null mutants properly localize rabphilin (Fig.…”

“…Association with rab3 requires the N-terminal cysteine-rich Zn 2ϩ -binding region (McKiernan et al, 1996;Stahl et al, 1996;Ostermeier and Brunger, 1999). It remains unclear whether rab3 recruits rabphilin to synaptic vesicles (Li et al, 1994;McKiernan et al, 1996;Stahl et al, 1996) or whether rabphilin also interacts with vesicles independently of rab3 Arribas et al, 1997).…”

“…Association with rab3 requires the N-terminal cysteine-rich Zn 2ϩ -binding region (McKiernan et al, 1996;Stahl et al, 1996;Ostermeier and Brunger, 1999). It remains unclear whether rab3 recruits rabphilin to synaptic vesicles (Li et al, 1994;McKiernan et al, 1996;Stahl et al, 1996) or whether rabphilin also interacts with vesicles independently of rab3 Arribas et al, 1997). The rabphilin C terminus contains two protein kinase C-like C2 domains that bind phospholipid in a Ca 2ϩ -dependent manner and that may play a role in membrane association McKiernan et al, 1996;Chung et al, 1998;Ubach et al, 1999).…”