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

The above results strongly argued for a role of the cytoplasmic domain in intracellular targeting of ERGIC-53, and to analyze this directly two CD4-ERGIC53 constructs were generated: IAT4C53, containing only the cytoplasmic domain, and IAT53C53 containing the cytoplasmic and the transmembrane domain of ERGIC-53 (Fig. 1) . Transfected COS cells were analyzed by a double immunofluorescence procedure which allowed differential staining of the cell surface and intracellular membranes ( Fig. 5 ; see Materials and Methods). Overexpressed CD4 showed strong surface staining under nonpermeabilized and a perinuclear intracellular staining under permeabilized conditions. The perinuclear staining most likely represents newly synthesized protein in transit through the Golgi as well as internalized CD4 concentrated in endosomes and/or lysosomes. Overexpressed CD4 chimera IAT53C53 and L4T4C53, on the other hand, showed no surface staining but a strong staining indicative of the ER: reticular intracellular staining with a nuclear ring (Fig. 5 , c-f). It is important to note that this staining is distinct from an E R G I C pattern (see also Fig. 8 B) . For L4T4C53, a few cells exhibited surface staining (insert in Fig. 5 , e and f). To confirm these results biochemically, transfected COS cells were pulse--chased and the immunoprecipitates were digested with endo H (data not shown). In contrast to wild-type CD4, the chimeras remained endo H sensitive after prolonged chase times indicating a pre-medial-Golgi localization, which is in line with the ER-staining observed in Fig. 5 . We conclude that the cytoplasmic domain of ERGIC-53 is sufficient for premedial-Golgi targeting.