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 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 beta-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 beta-COP. A quantitative binding assay was employed to measure recruitment of beta-COP to membrane 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 ribosylation factor, and protein kinase C-like activity. The ability to enhance beta-COP membrane binding was not limited to the peptide. Similarly, the addition of recombinant Rab2 protein to the assay promoted beta-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.
Tisdale
E J
EJ
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. etisdale@med.wayne.edu
Jackson
M R
MR
D000595:Amino Acid Sequence; D020756:Coatomer Protein; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism; D020693:rab2 GTP-Binding Protein
D020756:Coatomer Protein; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009281:Naphthalenes; D014759:Viral Envelope Proteins; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D020693:rab2 GTP-Binding Protein; C058819:calphostin C