Rab1 and Ca2+ are required for the fusion of carrier vesicles mediating endoplasmic reticulum to Golgi transport.

Abstract

Abstract. Members of the rab/YPTI/SEC4 gene family of small molecular weight GTPases play key roles in the regulation of vesicular traffic between compartments of the exocyfic pathway. Using immunoelectron microscopy, we demonstrate that a dominant negative rabla mutant, rabla(N124I), defective for guanine nucleotide binding in vitro, leads to the accumulation of vesicular stomatitis virus glycoprotein (VSV-G) in numerous pre-cis-Golgi vesicles and vesicular-tubular clusters containing rabl and B-COP, a subunit of the coatomer complex. Similar to previous observations . Cell. 76:841-852), VSV-G was concentrated nearly 5-1G-fold in vesicular carriers that aocumulate in the presence of the rabla(N124I) mutant. VSV-G containing vesicles and vesiculartubular clusters were also found to accumulate in the presence of a rabla effector domain peptide mimetic that inhibits endoplasmic reticulum to Golgi transport, as well as in the absence of Ca 2+. These results suggest that the combined action of a Ca2+-dependent prorein and conformational changes associated with the GTPase cycle of rabl are essential for a late targeting/fusion step controlling the delivery of vesicles to Golgi compartments.

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.

“…Previously, both mutants were found to be potent inhibitors of ER to Golgi and intra-Golgi traffic (14,42,45). By analogy to the equivalent mutation in H-ras (S17N) (24), the rabla(S25N) mutant is likely to bind in a competitive fashion to a tab-specific GEP and prevent the normal function of this protein in promoting GDP/GTP exchange and recruitment of wild-type rabl (42,44).…”

“…The first, rabla(S25N), is analogous to H-ras(S17N) which has a preferential affinity for GDP (24,42) and in the case of H-ras is growth inhibitory due to its sequestration of the rasspecific guanine nucleotide exchange protein (22,24). The second mutant, rabla(N124I) fails to bind either GDP or GTP at detectable levels in vitro, sinailar to the analogous transforming H-ras(Nll6D mutant (45,58). These bacterially expressed rab proteins lack posttranslational modifications-such as prenylation, acylation or methylafion-which are normally a prerequisite for the functional association of ras-related proteins with membranes (37).…”

“…The purification of histidine-ta88ed canine rabla, rabla(N1241), and rabla(S25N) protein from Escherichia coil cell lysates is described in detail elsewhere (42,45). For these experiments, rabla wild-type or mutant proteins were concentrated by ultrafiltrafion to a final concentration of ",0.3-0.5 mg/ml and stored in aliquots at -70°C.…”

“…For these experiments, recombinant wild-type and mutant rabla proteins were isolated from bacterial lysates after expression in E. coll. Nucleotide-binding properties of rabla proteins purified in this manner are described elsewhere (42,45). Two mutants were tested in our microinjection assay system.…”

“…The necessity for cycling of rab and other GTI'ases involved in vesicular traffic is underscored by the sensitivity of many vesicular transport steps to the introduction of nonhydrolyzable analogues of GTP into cell-free or permeabilized cell transport assays (8,41). In particular, mutational analysis of closely related rabla and rablb proteins (14, 45) and ARF1 (13) strongly implicate them in control of trafficking events from the ER to the Golgi apparatus and between early Golgi compartments (14,42,45).…”