State-specific assays to study biosynthetic cargo selection and role of SNAREs in export from the endoplasmic reticulum and delivery to the Golgi

To analyze the role of coat protein type II (COPII) coat components and tar geting and fusion factors in selective export from the endoplasmic reticulu m (ER) and transport to the Golgi, we have developed three novel, stage-spe cific assays. Cargo selection can be measured using a "stage 1 cargo captur e assay," in which ER microsomes are incubated in the presence of glutathio ne S-transferase (GST)-tagged Sar1 GTPase and purified Sec23/24 components to follow recruitment of biosynthetic cargo to prebudding complexes. This c argo recruitment assay can be followed by two sequential assays that measur e separately the budding of COPII-coated vesicles from ER microsomes (stage 2) and, finally, delivery of cargo-containing vesicles to the Golgi (stage 3). We show how these assays provide a means to identify the snap receptor (sNARE) protein rBet1 as an essential component that is not required for v esicle formation, but is required for vesicle targeting and fusion during E R-to-Golgi transport. In general, these assays provide an approach to chara cterize the biochemical basis for the recruitment of a wide variety of bios ynthetic cargo proteins to COPII vesicles and the role of different transpo rt components in the early secretory pathway of mammalian cells. (C) 2000 A cademic Press.