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

COS cells transfected with GM, L53T4C53 and L53T4R2AI0 were pulsed for five min with [35S]methionine and chased for up to 2 h. The immunoprecipitated mutant proteins were digested with endo H and the fraction of resistant protein determined (Fig. 4 a) . G M representing overexpressed wild-type ERGIC-53 showed ~10% endo H resistance after 1 h of chase (Fig. 4 a) . This value was identical to that obtained with an ERGIC-53 mutant containing the N-glycosylation site but no epitope tag (not shown). The replacement of the transmembrane domain in ERGIC-53 by CD4 (L53T4C53) did not change the acquisition of endo H resistance, suggesting that it is not required for targeting (Fig. 4 a) . The inverse construct with the ERGIC-53 transmembrane domain in a CD4 background IAT53C4 showed similar kinetics of endo H resistance (Fig. 4b) and comparable localization by immunofluorescence (not shown) as overexpressed wild type CD4. This indicated that the transmembrane domain of ERGIC-53 was not sufficient for the targeting of ERGIC-53. Substitution of both the transmembrane and the cytoplasmic domains (L53T4R2A10) resulted in ~50% endo H resistance after 1 h of chase (Fig. 4 a) . The lumenal domain of ER-GIC-53 is not sufficient for targeting.