LOCALIZATION, DYNAMICS, AND PROTEIN INTERACTIONS REVEAL DISTINCT ROLES FOR ER AND GOLGI SNARES

ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide-sensitive factor attachment p rotein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin , rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22 b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs), Ho wever, all of the SNAREs were found on both COPII- and COPI-coated mem branes, indicating that similar SNARE machinery directs both vesicle p athways. rsec22b and rbet1 do not appear beyond the first Golgi cister na, whereas syntaxin 5 and membrin penetrate deeply into the Golgi sta cks. Temperature shifts reveal that membrin, rsec22b, rbet1, and synta xin 5 are present together on membranes that rapidly recycle between p eripheral and Golgi-centric locations. GOS-28, on the other hand, main tains a fixed localization in the Golgi. By immunoprecipitation analys is, syntaxin 5 exists in at least two major subcomplexes: one containi ng syntaxin 5 (34-kD isoform) and GOS-28, and another containing synta xin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subc omplexes appear to involve direct interactions of each SNARE with synt axin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusio n interfaces: the fusion of ER-derived vesicles with VTCs, and the ass embly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform , membrin, and GOS-28 may function in intraGolgi transport.