Mutation of small, polar, and aromatic residues in TM2 of MARCH9 affects substrate down-regulation.
A, sequences of mutants analyzed in this figure. Red bold font indicates substituted amino acids. B–E, 293T cells stably expressing human CD4 were transduced with the indicated lentiviral constructs, and surface levels of HLA-A2 (B and D) and CD4 (C and E) were measured after 48 h of culture with or without dox as in Fig. 1. Dashed lines indicate mean substrate level remaining on dox-treated cells expressing control WT (lower) and inactive mutant S198A (upper) MARCH9. Unpaired t test: ns, not significant; *, p < 0.05; **, p < 0.02; ***, p < 0.001; ****, p < 0.0001.