RAB2 PROTEIN ENHANCES COATOMER RECRUITMENT TO PRE-GOLGI INTERMEDIATES

The Rab2 protein is a resident of pre-Golgi intermediates and required for vesicular transport in the early secretory pathway. We have previ ously shown that a peptide corresponding to the amino terminus of Rab2 (residues 2-14) arrests protein traffic prior to a rate-limiting even t in VSV-G movement through pre-GoIgi structures (Tisdale, E. J., and Balch, W. E. (1996) J. Biol, Chem. 271, 29372-29379). To determine the mechanism by which this peptide inhibits transport, we investigated t he effect of the Rab2 peptide on the distribution of the beta-COP subu nit of coatomer because COPI partially localizes to pre-Golgi intermed iates, We found that the peptide caused a dramatic change in the distr ibution of pre-Golgi intermediates containing beta-COP. A quantitative binding assay was employed to measure recruitment of beta-COP to memb rane when incubated with the Rab2 (13-mer). Peptide-treated microsomes showed a 25-70% increase in the level of membrane-associated beta-COP . The enhanced recruitment of coatomer to membrane was specific to the Rab2 (13-mer) and required guanosine 5'-3-O-(thio)triphosphate, ADP r ibosylation factor, and protein kinase C-like activity. The ability to enhance beta-COP membrane binding was not limited to the peptide. Sim ilarly, the addition of recombinant Rab2 protein to the assay promoted beta-COP membrane association. Our results suggest that the Rab2 pept ide causes the persistent recruitment of COPI to pre-Golgi intermediat es which ultimately arrests protein transport due to the inability of membranes to uncoat.