Exploring the consequences of redirecting an exocytic Rab onto endocytic vesicles.

Abstract

Bidirectional vesicular traffic links compartments along the exocytic and endocytic pathways. Rab GTPases have been implicated in specifying the direction of vesicular transport because anterograde vesicles are marked with a different Rab than retrograde vesicles. To explore this proposal, we sought to redirect an exocytic Rab, Sec4, onto endocytic vesicles by fusing the catalytic domain of the Sec4 GEF, Sec2, onto the CUE localization domain of Vps9, a GEF for the endocytic Rab, Ypt51. The Sec2GEF-GFP-CUE construct was found to localize to bright puncta predominantly near sites of polarized growth and this localization was strongly dependent upon the ability of the CUE domain to bind to the ubiquitin moieties added to the cytoplasmic tails of proteins destined for endocytic internalization. Sec4 and Sec4 effectors were recruited to these puncta with varying efficiency. The puncta appeared to consist of clusters of 80 nm vesicles and although the puncta are largely static, FRAP analysis suggests that traffic into and out of these clusters continues. Cells expressing Sec2GEF-GFP-CUE grew surprisingly well and secreted protein at near normal efficiency, implying that Golgi derived secretory vesicles were delivered to polarized sites of cell growth, where they tethered and fused with the plasma membrane despite the misdirection of Sec4 and its effectors. In total, the results suggest that while Rabs play a critical role in regulating vesicular transport, cells are remarkably tolerant of Rab misdirection.