Morphological and functional association of Sec22b/ERS-24 with the pre-Golgi intermediate compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular tran sport between the endoplasmic reticulum (ER) and the Golgi apparatus by fun ctioning as a V-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] atta chment protein receptor) of transport vesicles. Three mammalian proteins ho mologous to Sec22p have been identified and are referred to as Sec22a, Sec2 2b/ERS-24, and Sec22c, respectively. The existence of three homologous prot eins in mammalian cells calls for detailed cell biological and functional e xaminations of each individual protein. The epitope-tagged forms of all thr ee proteins have been shown to be primarily associated with the ER, althoug h functional examination has not been carefully performed for any one of th em. In this study, using antibodies specific for Sec22b/ERS-24, it is revea led that endogenous Sec22b/ERS-24 is associated with vesicular structures i n both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the en velope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route f rom the ER to the Golgi under normal, brefeldin A, or nocodazole-treated ce lls suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate co mpartment (IC). In a well-established semi-intact cell system that reconsti tutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER-Golgi transp ort at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER-Golgi tran sport only when they are added before the EGTA-sensitive stage. Transport o f VSVG accumulated in pre-Golgi IC by incubation at 15 degrees C is also in hibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported f rom the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. Ln the presence of antibodi es against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates , suggesting that Sec22b/ERS-24 functions at the level of the IC in ER-Golg i transport.