Abstract
Rab GTPases have been implicated in intracellular vesicle trafficking. Using the yeast two-hybrid screen, we have isolated a rat clone that interacts with Rab3A as well as with Rab1. The gene encodes a 20.6-kDa protein with two extensive hydrophobic domains and is broadly expressed in all tissues. This protein binds to prenylated Rab GTPases but not to other small Ras-like GTPases such as the Rho/Rac family. This prenylated Rab acceptor (PRA1) also binds specifically to the synaptic vesicle protein VAMP2 (or synaptobrevin II) but shows no affinity for VAMP1 or cellubrevin in both the yeast two-hybrid system and in vitro binding assays. This specificity resides, in part, in the proline-rich domain of VAMP2 as a chimera containing this domain of VAMP2 fused to VAMP1 is able to bind to PRA1. The transmembrane domain of VAMP2 is also essential as its deletion abolished binding to PRA1. Replacement of the deleted VAMP2 transmembrane domain by a CAAX prenylation signal can not restore binding to PRA1. This interaction is therefore distinct from that required for VAMP2 binding to either syntaxin or both syntaxin and SNAP-25. Deletion analysis on PRA1 indicates that the critical Rab- and VAMP2-interacting residues reside in two regions: the amino-terminal residues 30-54 and the extreme carboxyl-terminal domain. This dual Rab and VAMP2 binding characteristic suggests that PRA1 may serve to link these two protein families in the control of vesicle docking and fusion.
Highlights
The Rab family of GTPases plays a regulatory role in the intracellular transport of vesicles by cycling through GTP- and GDP-bound states [1, 2]
Since targeting or recycling of VAMP2 to synaptic vesicle is affected by mutations at residues 46 and 49 [44], we examined the effects of these mutations on PRA1 interaction
The PRA1 clone was isolated as a Rab3A-interacting clone in a yeast two-hybrid screen of a rat brain cDNA library
Summary
The Rab family of GTPases plays a regulatory role in the intracellular transport of vesicles by cycling through GTP- and GDP-bound states [1, 2]. The Amino- and Carboxyl-terminal Amino Acids in PRA1 Are Essential for Rab and VAMP2 Binding—To determine the domains within PRA1 responsible for binding to prenylated Rab GTPase and to VAMP2, deletion analysis was performed on PRA1. Deletion of the carboxyl-terminal 21 amino acids (PRA1(1–164)) and the second hydrophobic domain (PRA1(1– 110)) abolished interaction to both Rab3A and VAMP2 (Fig. 6).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
