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

To define the targeting signal(s) in the cytoplasmic domain of ERGIC-53, amino acids were serially truncated from the C O O H terminus. Surprisingly, deletion of up to seven amino acids, which eliminated the putative KKXX ER-retrieval signal, did not substantially change the rate of endo H resistance compared to wild type (Fig. 6, lines 2 , 3, 4, and 6). These results suggest that the inner five amino acids R S Q Q E adjacent to the transmembrane domain act Figure 5 . Double-immunofluorescence analysis of surface and intracellular CD4, 1_AT53C53 and L4T4C53. Transfected COS cells were cooled to 4°C and incubated with mAb 6D10 (IgG1), against CD4, followed by IgGl-specific goat antimouse-FITC to stain protein at the cell surface (b, d, and f). The cells were then fixed and permeabilized followed by an incubation with mAb HP2/6.1 (IgG2a), against CD4, and goat anti-mouse IgG2a-specific goat anti-mouse-TRITC staining the intracellular protein (a, c, and e). The DNA's used were CD4 (a and b), L4T53T53 (c and d) and L4T4C53 (e and Y3-Bar corresponds to 10 ~m in a, b, c, d and 22 ~m in e and f. as a targeting determinant. To exclude conformational artifacts due to truncation, which might slow down exit from the ER, we restored the authentic tail length by re-adding seven alanines to the R S Q Q E , and we deleted the KKXXmotif in L53T4C53. Both constructs showed equal endo H resistance and oligomerization equal to wild type, strongly arguing against a truncation artifact (Fig. 6 , lines 5 and 7). We conclude that the five residues R S Q Q E adjacent to the transmembrane domain constitute a targeting determinant. Figure 6 . The cytoplasmic domain of ERGIC-53 contains a KKXX-retrieval signal and a second targeting signal adjacent to the membrane. Endo H resistance was determined after 5 rain of pulse with [35S]methionine followed by 60 min of chase as described in Fig. 4 a. The mutations indicated were all constructed in a GM background, except mutation 5 that was constructed in L53T4C53 (see Fig. 1 ). The amino acid sequence of the cytoplasmic domain is given in the single letter code with construct 1 representing the wild-type sequence. The values given are means + SD of triplicate cultures treated in parallel. Surface appearance of the overexpressed proteins was analyzed by indirect immunofluorescence microscopy staining the cell surface with mAb 9El0 at 4°C before fixation and permeabilization as described in Fig. 2 . We next studied the role of the lysines in the cytoplasmic domain of ERGIC-53 (Fig. 6, line 9) . A construct with two correctly positioned lysines ( -3 / -4 from the COOH terminus) but lacking the terminal phenylalanines did not acquire endo H resistance and was not transported to the cell surface. This construct is significantly different from construct 1 (Fig. 6, wt) , containing the terminal phenylalanines and from construct 7 (Fig. 6 ) that contains only the RSQQE determinant. It also confirmed that transport of ERGIC-53 to the Golgi and the cell surface was not simply a question of enforced overexpression but an intrinsic property of the cytoplasmic domain.