Fig. 1. The FF transport motif of ERGIC-53 can be functionally substituted by other motifs. (A) C-terminal amino acid sequences of ERGIC-53 from different species. Positions relative to the C-terminus are indicated. (B) A schematic representation of ERGIC-53 constructs expressed in COS cells. All constructs have an N-glycosylation site (CHO) at position 61 and a c-myc epitope (Itin et al., 1995). The TMD is followed by the amino-acid sequence of the cytoplasmic tail, in which the two lysines at position -3 and -4 were replaced by alanines to prevent recycling. The -1 and -2 positions (XX) were mutated to the amino acids indicated in panels C to E. (C) Effect of double substitutions of the FF motif. COS cells were transfected with the indicated constructs and subjected to pulse-chase/endo H analysis using [³⁵S]-methionine. 60 minutes after the chase, the cells were lysed and ERGIC-53 constructs were immunoprecipitated with anti-myc. Immunoprecipitates were treated with endo H, separated by SDS-PAGE and analyzed by fluorography. The upper band represents the endo H-resistant and the lower band the endo H-sensitive form ERGIC-53. (D) Quantification of fluorograms shown in panel C. (E) Acquisition of endo H resistance of ERGIC-53 constructs with single motifs at the XX position. White bars in D an E represent values for the signalless reporter. Black bars represent values obtained with the FF construct. Mean values±s.d. of five independent experiments are shown.