FIGURE 3.

Effects of Cys substitution and subsequent thiol-reactive reagent treatment on residues in TM4. A, NANA transport activity (relative to Cys-less sialin) for each TM4 monocysteine substitution mutant. Transport of 30 nm ³H-NANA was measured after 5 min at pH 5.5. B, inhibition of monocysteine substitution mutants after NEM (filled bars) or MTSET (open bars) pretreatment. Only monocysteine substitution mutants for which NEM inhibited activity by greater than 15% are shown. The effects on the other mutants are presented in supplemental Fig. 2. Cells were pretreated in presence of 2 mm NEM at pH 7.5 or 2 mm MTSET at pH 5.5 for 5 min and transport then measured as in A. C, helical wheel plot of TM4. Circles indicate residues where cysteine substitution was associated with loss of >95% measurable transport. Dashed lines indicate residues where substitution led to markedly decreased expression (see supplemental Fig. 1). Triangles indicate positions where cysteine substitution led to NEM-sensitive transport with the filled triangle at Phe-179 indicating that substitution led to inhibition by MTSET as well as NEM.