The Rab5 Effector EEA1 Interacts Directly with Syntaxin-6*
Abstract
The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate intrans-Golgi network to endosome as well as in endocytic membrane traffic.
Footnotes
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↵* This work was supported by the Top Research Program, the Research Council of Norway, the Novo Nordisk Foundation, and the Norwegian Cancer Society.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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↵‡ To whom correspondence should be addressed. Tel.: +47-22934951; Fax: +47-22508692; E-mail: stenmark@ulrik.uio.no.
- Abbreviations:
- SNARE
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solubleN-ethylmaleimide-sensitive factor attachment protein receptor
- GST
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glutathione S-transferase
- MBP
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maltose-binding protein
- TGN
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trans-Golgi network
- PBS
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phosphate-buffered saline
- PAGE
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polyacrylamide gel electrophoresis
- PtdIns-(3)P
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phosphatidylinositol 3-phosphate
- DTT
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dithiothreitol
- GTPYS
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guanosine 5′-3-O-(thio)triphosphate
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- Received June 2, 1999.
- Revision received July 22, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
