THE MAMMALIAN PROTEIN (RBET1) HOMOLOGOUS TO YEAST BET1P IS PRIMARILY ASSOCIATED WITH THE PRE-GOLGI INTERMEDIATE COMPARTMENT AND IS INVOLVEDIN VESICULAR TRANSPORT FROM THE ENDOPLASMIC-RETICULUM TO THE GOLGI-APPARATUS

Yeast Bet1p participates in vesicular transport from the endoplasmic r eticulum to the Golgi apparatus and functions as a soluble N-ethylmale imide-sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Be t1p was recently identified, and it was concluded that rbet1 is associ ated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study usin g rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi app aratus as marked by the Golgi mannosidase II in normal rat kidney cell s. Rather, rbet1 is predominantly associated with vesicular spotty str uctures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL r eceptor and ERGIC-53, which are known to be enriched in the intermedia te compartment. When the Golgi apparatus is fragmented by nocodazole t reatment: a significant portion of rbet1 is not colocalized with struc tures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered b y preincubation of cells at 15 degrees C. However, upon warming up fro m 15 to 37 degrees C, rbet1 concentrates into the peri-Golgi region. F urthermore, rbet1 colocalizes with vesicular stomatitis virus G-protei n en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincu bation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the act ual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These result s suggest that rbet1 may be involved in the docking process of ER deri ved vesicles with the cis-Golgi membrane.