Roles for α₂p24 and COPI in Endoplasmic Reticulum Cargo Exit Site Formation

A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg²⁺ATP and Mg²⁺GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins α₂p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of α₂p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of α₂p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTPγS, inhibition by Brefeldin A (BFA), or depletion of β-COP from cytosol. Therefore, the p24 family member, α₂p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the eneration of ER cargo exit sites.