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

Besides suppressing the inner targeting determinant do the COOH-terminal phenylalanines also influence the dilysine signal? Construct 9 in Fig. 6 , which had the terminal phenylalanines replaced by alanines in the wild-type cytoplasmic domain, was not transported to the medial-Golgi after 5 h of chase (Fig. 7, line 2) . This result, however, did not allow us to conclude that the terminal phenylalanines influence the di-lysine motif since in that construct the inner targeting determinant was not suppressed and could have contributed to the intracellular targeting efficiency (compare Fig. 7, lines 1 and 2) . However, substitution of the phenylalanines in R R A A A A A A K K F F by atanines enabled us to determine the influence of the phenylalanines on the di-lysine motif. The resulting construct with the cytoplasmic amino acid sequence RRAAAAAAK-KAA (Fig. 7, line 4) showed no significant ER to medial-Golgi transport after 5 h of chase comparable to RSQ-Q E A A A K K A A (Fig. 7, line 2) . Both constructs showed expression levels identical to GM and displayed no surface staining by immunofluorescence microscopy (data not shown).