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. INTRODUCTIONMammalian cells endocytose a variety of molecules. Some of them escape from the lysosomal degradative pathway and are instead delivered to the Golgi apparatus. Examples of these are some protein toxins such as cholera toxin, Shiga toxin, and ricin (Sandvig and van Deurs, 2002) as well as some endogenous proteins such as TGN38 (Ghosh et al., 1998;Mallet and Maxfield, 1999), mannose 6-phosphate receptor (Goda and Pfeffer, 1988), furin (Mallet and Maxfield, 1999), GLUT4 (Shewan et al., 2003), and some glycosylphosphatidylinositol (GPI)-anchored proteins (Nichols et al., 2001;Nichols, 2002). To date, several retrograde transport pathways in the endocytic route to the trans-Golgi network (TGN) have been identified. These include a well-studied pathway from the late endosome to the TGN taken by mannose 6-phosphate receptors (Goda and Pfeffer, 1988) and furin (Mallet and Maxfield, 1999), and a newly discovered direct pathway from the early/recycling endosome to the TGN taken by TGN38 (Ghosh et al., 1998;Mallet and Maxfield, 1999), GLUT4 (Shewan et al., 2003), and exogenously added Shiga toxin B subunit (Mallard et al., 1998). Similar transport from the late endosome (prevacuolar compartment) and the early/recycling endosome (post-Golgi compartment) to the TGN (late Golgi) has been described in the yeast Saccharomyces cerevisiae (Bensen et al., 2001;Siniossoglou et al., 2001). Recently, GPI-anchored green fluorescent protein (GFP), CD59, and a fraction of cholera toxin B subunit have been found to accumulate in a discrete population of endosomes en route to the Golgi apparatus (Nichols, 2002). These endosomes are devoid of markers for classical early and recycling endosomes, but they do...