Rab11BP, a putative effector of Rab11, may regulate vesicular transport through an interaction with AP-1 adaptor.

摘要

Rab11BP is a 912 as rab11 binding protein that is mainly cytosolic and interacts with the GTP-bound form of rab11, a rab known to regulate vesicular transport in the endocytic pathway. In vitro binding of rab11 to rab11BP requires partial denaturation of the latter to expose an internal binding site (aa 334–504) that is apparently masked by the C-terminal region of rab11BP, which contains six WD40 repeats. Within the cell, rab11BP must undergo a conformational change that enables it to bind rab11 since, when coexpressed in transfected cells, the two proteins form abundant membrane-associated complexes. I have searched for proteins that interact with rab11BP and may alter its conformation, serve to facilitate its association with membranes, and/or function as its downstream effectors. In a yeast two-hybrid screen I identified the AP-1 adaptor σ1 subunit as a rab11BP interacting protein. GST pull-down experiments in cotransfected cells confirmed that the interaction takes place in vivo and localized the σ1 binding sites in rab11BP to the first two WD40 repeats (aa 504–554 and 604–643). I also demonstrated that rab11BP can interact with 61 subunits incorporated into AP-1 complexes. Confocal microscopy showed that GST-rab11BP was partially colocalized with endogenous AP-1 complexes in TGN and endosomal compartments. In an assay for transport of newly synthesized VSVG-GFP from the TGN to the cell surface, the fragments comprising as 504–554 or 604–643, but not the full-length rab11BP or a WD40 repeat that does not bind to σ1, inhibited VSV-G transport. I also found in cotransfected cells that rab11BP forms homodimers through an interaction mediated by the third WD40 repeat (aa 644–688). Our work is now directed to determining whether the interaction of σ1 with rab11BP reflects a mechanism for the recruitment of rab11 to a budding vesicle containing AP-1, or whether a rab11BP-rab11 complex first formed on endosomal membranes brings AP-1 to the site of budding. In any case, dissociation of the homodimers may be required for rab11BP to bind rab11-GTP.