A Rab2 mutant with impaired GTPase activity stimulates vesicle formation from pre-Golgi intermediates.
Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound-activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of beta-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated beta-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with beta-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, beta-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15 degrees C. These data suggest that the Rab2 protein plays a role in the low-temperature-sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.
Tisdale
E J
EJ
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. etisdale@med.wayne.edu
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line / Q000187:drug effects; D018699:Coated Vesicles / Q000378:metabolism*; D020756:Coatomer Protein; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D037241:Mannose-Binding Lectins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009154:Mutation; D009281:Naphthalenes / Q000494:pharmacology; D051381:Rats; D014759:Viral Envelope Proteins / Q000378:metabolism; D020693:rab2 GTP-Binding Protein
D020756:Coatomer Protein; D004791:Enzyme Inhibitors; C028391:G protein, vesicular stomatitis virus; C501458:Lman1 protein, rat; D037241:Mannose-Binding Lectins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009281:Naphthalenes; D014759:Viral Envelope Proteins; C061465:calphostin complex; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020693:rab2 GTP-Binding Protein