MSS4 DOES NOT FUNCTION AS AN EXCHANGE FACTOR FOR RAB IN ENDOPLASMIC-RETICULUM TO GOLGI TRANSPORT

Mss4 and its yeast homologue, Dss4, have been proposed to function as guanine nucleotide exchange factors (GEFs) for a subset of Rab protein s in the secretory pathway. We have previously shown that Rab1A mutant s defective in GTP-binding potently inhibit endoplasmic reticulum to G olgi transport, presumably by sequestering an unknown GEF regulating i ts function. We now demonstrate that these mutants stably associate wi th Mss4 both in vivo and in vitro and that Mss4 effectively neutralize s the inhibitory activity of the Rab1A mutants. An equivalent Rab3A mu tant (Rab3A[N135I]), a Rab protein specifically involved in regulated secretion at the cell surface, associates with Mss4 as efficiently as the Rab1A[N124I] mutant. Although Rab3A[N135I] prevents the ability of Mss4 to neutralize the inhibitory effects of Rab1A mutants on transpo rt, it has no effect on Rab1 function or endoplasmic reticulum to Golg i transport. Furthermore, quantitative immunodepletion of Mss4 fails t o inhibit transport in vitro. We conclude that Mss4 and its yeast homo logue, Dss4, are not GEFs mediating activation of Rab, but rather, the y interact with the transient guanine nucleotide-free state, defining a new class of Ras-superfamily GTPase effectors that function as guani ne nucleotide-free chaperones (GFCs).