Cargo Selection by the COPII Budding Machinery during Export from the ER

TLDR

Cargo is selectively exported from the ER in COPII vesicles. The study aimed to dissect COPII’s role in selective ER export by purifying mammalian COPII components from rat liver cytosol and examining their involvement in cargo selection. The authors used GST‑tagged Sar1 or Sec23 recruited to ER microsomes, then solubilized and reisolated the tagged subunits with glutathione‑Sepharose beads to probe cargo–coat interactions. The purified Sec23–24 complex (85‑kD Sec23 and 120‑kD Sec24) is the minimal cytosolic component recruited after Sar1 activation, but Sec13–31 is required for full budding; activated Sar1 with Sec23 or Sec23–24 efficiently recruits the type‑1 transmembrane cargo VSV‑G into a detergent‑soluble prebudding complex that excludes ER residents, and addition of Sec13–31 drives its export into COPII vesicles, demonstrating that cargo sorting occurs on the ER membrane before budding and depends on GTPase‑dependent coat recruitment.

Abstract

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.