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

To assess whether the KKFF sequence is a functional dilysine ER-retrieval signal, we tested two criteria: lysine dependence and position dependence from the COOH terminus (Jackson et al., 1990) . Both, the lysine dependence tested by substitution with arginines (Fig 6, line 15 ) and the position dependence from the COOH terminus tested by addition of three alanines to the COOH terminus (Fig. 6 , lines 16 and 17, and compare line 9 to 18) identified the KKFF-tetrapeptide as a di-lysine ER-retrieval signal. We conclude that the cytoplasmic domain of ERGIC-53 contains three targeting determinants: a di-lysine ER-retrieval signal, a RSQQE determinant adjacent to the membrane and two COOH-terminal phenylalanines which influence the RSQQE determinant. It is important to note that the overall folding of all mutants generated in a GM background was controlled by comparing their kinetics of oli- Figure 7 . The retrieval capacity of the di-lysine signal is reduced by the phenylalanines. Endo H resistance was determined as in Fig. 5 . Chase time was 5 h. All constructs were in a GM background containing the indicated cytoplasmic domains. Construct 1 corresponds to GM, and construct 2 is identical to construct 9 in Fig. 5 . The values given are means -+ SD of triplicate cultures treated in parallel.