IMMUNOISOLATION AND CHARACTERIZATION OF A SUBDOMAIN OF THE ENDOPLASMIC-RETICULUM THAT CONCENTRATES PROTEINS INVOLVED IN COPII VESICLE BIOGENESIS

Rubella virus E1 glycoprotein normally complexes with E2 in the endopl asmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex. In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre -Golgi compartment whose morphology and biochemical properties are dis tinct from to hypertrophied ER exit sites that have expanded in respon se to overexpression of E1. In the present study we constructed BHK ce lls stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by ce ll fractionation and immunoisolation. Double label indirect immunofluo rescence in cells and immunoblotting of immunoisolated tubular network s revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment. Furthermore, budding struc tures were evident in these membrane profiles, and a highly abundant b ut unknown 65-kDa protein was also present. By comparison, marker prot eins of the rough ER, Golgi, and COPI vesicles were not enriched in th ese membranes. These results demonstrate that the composition of the t ubular networks corresponds to that expected of ER exit sites. Accordi ngly, we propose the name SEREC (smooth ER exit compartment) for this structure.