A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates
10.1091/mbc.10.6.1837
Crossref journal-article
American Society for Cell Biology (ASCB)
Molecular Biology of the Cell (1076)
Abstract

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 β-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated β-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with β-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, β-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°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.

Bibliography

Tisdale, E. J. (1999). A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates. Molecular Biology of the Cell, 10(6), 1837–1849.

Authors 1
  1. Ellen J. Tisdale (first)
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Dates
Type When
Created 12 years, 1 month ago (July 2, 2013, 7:32 p.m.)
Deposited 3 years, 6 months ago (Feb. 26, 2022, 9:39 p.m.)
Indexed 2 months, 3 weeks ago (June 4, 2025, 5:31 a.m.)
Issued 26 years, 2 months ago (June 1, 1999)
Published 26 years, 2 months ago (June 1, 1999)
Published Print 26 years, 2 months ago (June 1, 1999)
Funders 0

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@article{Tisdale_1999, title={A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates}, volume={10}, ISSN={1939-4586}, url={http://dx.doi.org/10.1091/mbc.10.6.1837}, DOI={10.1091/mbc.10.6.1837}, number={6}, journal={Molecular Biology of the Cell}, publisher={American Society for Cell Biology (ASCB)}, author={Tisdale, Ellen J.}, editor={Pfeffer, Suzanne R.}, year={1999}, month=jun, pages={1837–1849} }