Fig. 4. Polar and aromatic residues in the TMD are required for efficient transport. (A) Replacement of ERGIC-53's TMD slows transport. The TMD of ERGIC-53 (T53) in GM constructs with (+) or without (–) the FF motif was substituted with different amino acid repeats with a total length of 18 residues. Constructs were expressed in COS cells and transport was probed by pulse-chase/endo H (Fig. 1). Black bars: GMA₇ (–) and GMA₅FF (+) constructs with ERGIC-53's TMD (T53). Grey, white and hatched bars: constructs with TMD substitutions as indicated. Results are mean±s.e.m. of at least three independent experiments. (B) Representation of ERGIC-53's TMD as helical wheel. Linear sequence of TMD (residues 481-498) of human ERGIC-53 is given below the wheel. Polar amino acids and glycine 494 are in italics. Polar and aromatic residues facing one side of TMD helix are in bold. Residues influencing transport of GMA₅FF are encircled in the helical wheel and marked by arrowheads in the linear sequence. (C) Transport of TMD mutants (fluorogram). COS cells were transfected with GM constructs containing either wild-type TMD (lane 1, GMA₇; lane 2, GMA₅FF) or amino acid substitutions in TMD of GMA₅FF (lanes 3 to 6 as indicated in D). 42 hours after transfection, cells were subjected to pulse-chase/endo H analysis. (D) Quantification of fluorograms including that shown in C. Results are mean±s.e.m. of at least three independent experiments. Grey bars, GMA₅FF constructs with substitutions in the TMD as indicated; white and black bars, GMA₇ (wt–) and GMA₅FF (wt+) with wild-type TMD. *Statistical significance to bar 2 (P<0.05, Student's t-test).