Rab2 Protein Enhances Coatomer Recruitment to Pre-Golgi Intermediates*
- From the ‡Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201 and the R. W. Johnson Pharmaceutical Research Institute, San Diego, California 92121
Abstract
The Rab2 protein is a resident of pre-Golgi intermediates and required for vesicular transport in the early secretory pathway. We have previously shown that a peptide corresponding to the amino terminus of Rab2 (residues 2–14) arrests protein traffic prior to a rate-limiting event in VSV-G movement through pre-Golgi 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 the effect of the Rab2 peptide on the distribution of the β-COP subunit of coatomer because COPI partially localizes to pre-Golgi intermediates. We found that the peptide caused a dramatic change in the distribution of pre-Golgi intermediates containing β-COP. A quantitative binding assay was employed to measure recruitment of β-COP to membrane when incubated with the Rab2 (13-mer). Peptide-treated microsomes showed a 25–70% increase in the level of membrane-associated β-COP. The enhanced recruitment of coatomer to membrane was specific to the Rab2 (13-mer) and required guanosine 5′-3-O-(thio)triphosphate, ADP ribosylation factor, and protein kinase C-like activity. The ability to enhance β-COP membrane binding was not limited to the peptide. Similarly, the addition of recombinant Rab2 protein to the assay promoted β-COP membrane association. Our results suggest that the Rab2 peptide causes the persistent recruitment of COPI to pre-Golgi intermediates which ultimately arrests protein transport due to the inability of membranes to uncoat.
Footnotes
-
↵* This work was supported in part by American Cancer Society Research Grant ACS IN-162 (to E. J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵§ To whom correspondence should be addressed. E-mail:etisdale{at}med.wayne.edu; Tel.: 313-577-1007; Fax: 313-577-6739.
-
↵1 The abbreviations used are: ER, endoplasmic reticulum; ARF, ADP-ribosylation factor; endo H, endoglycosidase H; PBS, phosphate-buffered saline; PKC, protein kinase C; VTC, vesicular tubular clusters; GTPγS, guanosine 5′-3-O-(thio)triphosphate; HRP, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis.
-
- Received February 11, 1998.
- Revision received April 2, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
