GUANINE-NUCLEOTIDE DISSOCIATION INHIBITOR IS ESSENTIAL FOR RAB1 FUNCTION IN BUDDING FROM THE ENDOPLASMIC-RETICULUM AND TRANSPORT THROUGH THE GOLGI STACK

The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medi al compartments of the Golgi stack. In the present study, we examine t he role of guanine nucleotide dissociation inhibitor (GDI) in regulati ng the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess G DI rapidly (t(1/2) < 30 s) extracted Rab1 from membranes, inhibiting v esicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractiona tes with a molecular mass of similar to 80 kD in the form of a GDI-Rab 1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, sup plementation of depleted cytosol with a GDI-Rab1 complex prepared in v itro from recombinant forms of Rab1 and GDI efficiently restored expor t from the ER, and transport through the Golgi stack. These results pr ovide evidence that a cytosolic GDI-Rab1 complex is required for the f ormation of non-clathrin-coated vesicles mediating transport through t he secretory pathway.