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

As the endo H assay cannot monitor localization within the ER-ERGIC-c/s-Golgi cycle we used immunofluorescence microscopy of cells expressing moderate levels of protein to distinguish ER from ERGIC and Golgi. High expression levels lead to saturation of the recycling pathway and result in staining of the cell surface and the endosomal system, a condition that renders a morphological analysis impossible (see also Fig. 2) . To determine the role of the cytoplasmic targeting signals in maintaining ERGIC-53 within the ER-ERGIC-cis-Golgi cycle, COS cells were transfected with the cDNAs corresponding to the constructs in Fig. 7 with the cytoplasmic domains: RSQ-QEAAAKKFF (wild type), RSQQEAAAKKAA, RRA-AAAAAKKFF, and RRAAAAAAKKAA. 16 h after transfection, corresponding to a threefold increased expression of mutant versus endogenous ERGIC-53 (data not shown), mutant protein was selectively visualized with the mAb against the c-myc epitope. Overexpression of GM with the cytoplasmic sequence RSQQEAAAKKFF lead to the characteristic concentrated perinuclear pattern (Fig. 8 A, a) observed for endogenous ERGIC-53 (Fig. 8 B, b) , whereas substitution of the terminal phenylalanines by alanines (RSQQEAAAKKAA) lead to a typical ER pattern with a pronounced staining of the nuclear envelope and less concentration in a perinuclear area (Fig. 8 A, b) . This finding emphasizes the importance of the phenylalanines in targeting. Proteins terminating in RRAAAA- periphery (Fig. 8 A, c and d) . The finding that neither of the two ER-retrieval signals alone was capable of mediating wild-type targeting showed the requirement for the RSQQE determinant for targeting. Therefore, ERGIC-53 localization requires all three features in the cytoplasmic domain of ERGIC-53: a di-lysine ER-retrieval signal, a RSQQE targeting determinant and two COOH-terminal phenylalanines modulating both targeting determinants. That the different mutants show differences in their distribution also argues against binding of the mutants to calnexin or even endogenous ERGIC-53 via their carbohydrate moiety as an explanation for their targeting within the ER-ERGIC-cis-Golgi cycle.