Roles for alpha(2)p24 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 descri bed. The first step is mediated by the hydrolysis of Mg(2+)ATP and Mg(2+)GT P, leading to the formation of a transitional ER (tER) with the soluble car go albumin, transferrin, and the ER-to-Golgi recycling membrane proteins al pha 2p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enri ched therein. Upon further incubation (step two) with cytosol and mixed nuc leotides, interconnecting smooth ER tubules within tER transforms into vesi cular tubular clusters (VTCs). The cytosolic domain of alpha(2)p24 and cyto solic COPI coatomer affect VTC formation. This is deduced from the effect o f antibodies to the COOH-terminal tail of alpha(2)p24, but not of antibodie s to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTP gamma S, inhibition by Brefeldin A (BFA), or depletion of beta-COP from cytosol. Therefore, the p24 family member, alpha(2)p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs an d the generation of ER cargo exit sites.