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J. Cell Biol.,
Volume 141, Number 1, April 6, 1998 61-70
Department of Cell Biology and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
Cargo is selectively exported from the ER in
COPII vesicles. To analyze the role of COPII in selective transport from the ER, we have purified components of the mammalian COPII complex from rat liver
cytosol and then analyzed their role in cargo selection and ER export. The purified mammalian Sec23-24
complex is composed of an 85-kD (Sec23) protein and a
120-kD (Sec24) protein. Although the Sec23-24 complex or the monomeric Sec23 subunit were found to be
the minimal cytosolic components recruited to membranes after the activation of Sar1, the addition of the
mammalian Sec13-31 complex is required to complete
budding. To define possible protein interactions between cargo and coat components, we recruited either glutathione-S-transferase (GST)-tagged Sar1 or GST-
Sec23 to ER microsomes. Subsequently, we solubilized
and reisolated the tagged subunits using glutathione-Sepharose beads to probe for interactions with cargo.
We find that activated Sar1 in combination with either Sec23 or the Sec23-24 complex is necessary and sufficient to recover with high efficiency the type 1 transmembrane cargo protein vesicular stomatitis virus glycoprotein in a detergent-soluble prebudding protein
complex that excludes ER resident proteins. Supplementing these minimal cargo recruitment conditions
with the mammalian Sec13-31 complex leads to export
of the selected cargo into COPII vesicles. The ability of
cargo to interact with a partial COPII coat demonstrates that these proteins initiate cargo sorting on the
ER membrane before budding and establishes the role
of GTPase-dependent coat recruitment in cargo selection.
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