A Putative Heterotrimeric G Protein Inhibits the Fusion of COPI-coated Vesicles
SEGREGATION OF HETEROTRIMERIC G PROTEINS FROM COPI-COATED VESICLES*
- J. Bernd Helms‡§,
- Désiré Helms-Brons‡,
- Britta Brügger‡,
- Ioannis Gkantiragas‡,
- Heike Eberle‡,
- Walter Nickel‡,
- Bernd Nürnberg¶,
- Hans-Hermann Gerdes‖ and
- Felix T. Wieland‡
- From the ‡Biochemie-Zentrum Heidelberg (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg,¶Freie Universitaet Berlin, Universitätsklinikum Rudolf Virchow, Institut für Pharmakologie, Thielallee 67-73, 14195 Berlin, and ‖Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
Abstract
Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4 −) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4 −-sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.
Footnotes
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↵* This work was supported by a grant from the Deutsche Forschungsgemeinschaft and of the Human Frontier Science Program Organization.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.
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↵§ Recipient of a fellowship from the Alexander von Humboldt Foundation. To whom correspondence should be addressed. Tel.: 49-6221-544688; Fax: 49-6221-544366; E-mail:Helms{at}urz.uni-heidelberg.de.
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↵1 The abbreviations used are: G protein, heterotrimeric GTP-binding protein; CHO, Chinese hamster ovary; NRK, normal rat kidney; PC, phosphatidylcholine; VSV, vesicular stomatitis virus; NEM, N-ethylmaleimide; ARF, ADP-ribosylation factor; GTPγS, guanosine 5′-O-(thiotriphosphate); hCgB-GFP, green fluorescent protein-tagged human chromogranin B; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; NSF, NEM-sensitive factor.
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↵2 B. Brügger, unpublished data.
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- Received December 8, 1997.
- Revision received February 17, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
