RAB8, A SMALL GTPASE INVOLVED IN VESICULAR TRAFFIC BETWEEN THE TGN AND THE BASOLATERAL PLASMA-MEMBRANE

Small GTP-binding proteins of the rab family have been implicated as r egulators of membrane traffic along the biosynthetic and endocytic pat hways in eukaryotic cells. We have investigated the localization and f unction of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to th e Golgi region, vesicular structures, and to the basolateral plasma me mbrane. Two-dimensional gel electrophoresis showed that rab8p was high ly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles tr ansporting the hemagglutinin protein (HA) of influenza virus to the ap ical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hyper-variable COOH-terminal region of rab 8, while transport of the influenza HA from the TGN to the apical surf ace and ER to Golgi transport were unaffected. We conclude that rab8 p lays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.