Stage-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 targeting and fusion factors in selective export from the endoplasmic reticulum (ER) and transport to the Golgi, we have developed three novel, stage-specific assays. Cargo selection can be measured using a "stage 1 cargo capture assay," in which ER microsomes are incubated in the presence of glutathione S-transferase (GST)-tagged Sar1 GTPase and purified Sec23/24 components to follow recruitment of biosynthetic cargo to prebudding complexes. This cargo recruitment assay can be followed by two sequential assays that measure 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 vesicle formation, but is required for vesicle targeting and fusion during ER-to-Golgi transport. In general, these assays provide an approach to characterize the biochemical basis for the recruitment of a wide variety of biosynthetic cargo proteins to COPII vesicles and the role of different transport components in the early secretory pathway of mammalian cells.
Allan
B B
BB
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Weissman
J
J
Aridor
M
M
Moyer
B
B
Chen
C D
CD
Yoo
J S
JS
Balch
W E
WE
D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins / Q000378:metabolism
D002352:Carrier Proteins; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010750:Phosphoproteins; C118289:SEC24 protein, S cerevisiae; C106113:SEC31 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae