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Vol. 15, Issue 9, 4011-4022, September 2004
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* Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Singapore;
Department of Biochemistry and Neurobiology Program, National University of Singapore, Singapore 117597, Singapore;
Molecular Mechanisms of Intracellular Transport, Unité Mixte Recherche 144 Curie/Centre National de la Recherche Scientifique, Institut Curie, F-75248 Paris Cedex 05, France; and
Traffic and Signaling Laboratories, Unité Mixte Recherche 144 Curie/Centre National de la Recherche Scientifique, Institut Curie, F-75248 Paris Cedex 05, France
Submitted December 9, 2003;
Revised May 12, 2004;
Accepted May 13, 2004
Monitoring Editor: Jean Gruenberg
An in vitro transport assay, established with a modified Shiga toxin B subunit (STxB) as a marker, has proved to be useful for the study of transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN). Here, we modified this assay to test antibodies to all known soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that have been shown to localize in the Golgi and found that syntaxin 5, GS28, Ykt6, and GS15 antibodies specifically inhibited STxB transport. Because syntaxin 5, GS28, Ykt6, and GS15 exist as a unique SNARE complex, our observation indicates that these four SNAREs function as a complex in EE/RE-TGN transport. The importance of GS15 in EE/RE-TGN transport was further demonstrated by a block in recombinant STxB transport in HeLa cells when GS15 expression was knocked down by its small interfering iRNA. Morphological analyses showed that some GS15 and Ykt6 were redistributed from the Golgi to the endosomes when the recycling endosome was perturbed by SNX3-overexpression, suggesting that GS15 and Ykt6 might cycle between the endosomes and the Golgi apparatus. Further studies indicated that syntaxin 5 and syntaxin 16 exerted their role in EE/RE-TGN transport in an additive manner. The kinetics of inhibition exhibited by syntaxin 16 and syntaxin 5 antibodies is similar.
Abbreviations used: EE/RE-TGN, early/recycling endosomes to trans-Golgi network; STxB, recombinant Shiga Toxin B subunit.
Online version of this article contains supporting material. Online version is available at www.molbiolcell.org.
|| Corresponding author. E-mail address: mcbhwj{at}imcb.a-star.edu.sg.
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