A Rab2 mutant with impaired GTPase activity stimulates vesicle formation from pre-Golgi intermediates

• Published in: Molecular biology of the cell 1999-06, Vol.10 (6), p.1837-1849
• Main Author: Tisdale, E J
• Format: Article
• Language: English
• Subjects: Animals; Biological Transport - drug effects; Cell Line - drug effects; Coated Vesicles - metabolism; Coatomer Protein; Dose-Response Relationship, Drug; Endoplasmic Reticulum - metabolism; Enzyme Inhibitors - pharmacology; Golgi Apparatus - metabolism; Golgi Apparatus - ultrastructure; GTP Phosphohydrolases - metabolism; GTP-Binding Proteins - genetics; GTP-Binding Proteins - isolation & purification; GTP-Binding Proteins - metabolism; Mannose-Binding Lectins; Membrane Glycoproteins; Membrane Proteins - metabolism; Microtubule-Associated Proteins - metabolism; Mutation; Naphthalenes - pharmacology; rab2 GTP-Binding Protein; Rats; Viral Envelope Proteins - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.10.6.1837

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.