Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway

To determine if ERGIC-53 (GM) behaves identical to overexpressed wild-type ERGIC-53, COS-1 cells were transiently transfected with the corresponding DNAs and analyzed by pulse-labeling with [35S]methionine followed by immunoprecipitation, or by indirect immunofluorescence microscopy. The introduced c-myc epitope tag was recognized by the epitope-specific mAb 9E10 (Evan et al., 1985) both in indirect immunofluorescence experiments (Fig. 2 , e and f) and immunoprecipitation (Fig. 3) . In pulse-chase experiments GM and overexpressed wild-type ERGIC-53 displayed virtually identical expression levels and oligomerization (Fig. 3) , and the localization revealed by immunofluorescence microscopy indicates that the modifications did not alter the intracellular distribution compared to overexpressed wild type (Fig. 2 ). However, both overexpressed proteins showed a staining of the cell periphery (Schindler et al., 1993) and T4 comprises the hydrophobic residues 375-395 of human CD4 (Maddon et al., 1985) . typical for ER, and led to a comparable fraction of transfected ceils exhibiting surface staining (compare Fig. 2 , c and d with e and f). Cells expressing identical levels of ERGIC-53 or GM bound equal amounts of radio-iodinated mAb to ERGIC-53 suggesting identical loss of both proteins from the ER-ERGIC-cis-Golgi recycling pathway (data not shown). After immunoprecipitation of GM with mAb 9El0 against the c-myc epitope, digestion with endo H confirmed that the introduced N-glycosylation consensus site was glycosylated and that the oligomerization state of GM was identical to that of ERGIC-53 (Fig. 3) . The kinetics of oligomerization of endogenous ERGIC-53 in nontransfected cells and that of overexpressed GM or ERGIC-53 in transfected cells was identical (not shown), indicating that the overall folding of GM is indistinguishable from ERGIC-53, and overexpressed ERGIC-53 and GM showed the same half-life in pulsechase experiments (not shown). In Fig. 3 hardly any endogenous ERGIC-53 is coprecipitated with GM demonstrating that disulfide-linked heterooligomers between GM and endogenous ERGIC-53 should not influence the interpretation of a mutational analysis. We conclude that overexpression of the GM construct in COS-1 cells fulfills the criteria required for a mutational analysis of the targeting determinant(s) of ERGIC-53.