Figure 3.

Serine 198 in the first predicted TM domain of MARCH9 is absolutely required for substrate down-regulation. A, sequences of mutants analyzed in this figure. Red bold font indicates substituted amino acids. Vertical box highlights the position of Ser-198. 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. Serine-to-alanine substitutions were examined in blocks of three or four (3S-A and 4S-A mutants; B and C) and separately for the single-serine mutants as indicated (D and E). Dashed lines indicate mean substrate level remaining on dox-treated cells expressing control WT (lower) and RING mutant W143A (upper) MARCH9. Unpaired t test: ns, not significant. F, Western blot analysis of MARCH9 WT and inactive mutant expression, performed as in Fig. 2E. + marks the position of a cellular product detected by SA-HRP; arrow marks the position of MARCH9 protein. Table shows expression normalized to GAPDH loading control and relative to MARCH9-WT, quantitated by densitometry.