FIGURE 3.

Glucose transport activities of single-Cys mutants and effects of pCMBS. A and B, each residue in TM7 of Cys-less Hxt7 (C-less) was individually replaced with cysteine to yield 21 mutants (L325C to Y345C). KY73 cells harboring plasmids encoding each mutant were grown to log phase at 30 °C in SMal(ura) medium, after which glucose transport activity was measured for 5 s at 30 °C with 0.1 mm d-glucose as substrate. Transport activity was normalized by cell number (A) or was normalized by the expression level of each mutant as determined by quantitative immunoblot analysis and then expressed relative to the value for Cys-less Hxt7 (B). Data are means ± S.E. (n = 4–6). C, KY73 cells expressing individual single-Cys mutants were incubated in the absence or presence of 0.5 mm pCMBS for 5 min at 30 °C before measurement of transport activity with 0.1 mm d-glucose as substrate. Data are expressed as percentage inhibition of transport activity by pCMBS and are means ± S.E. (n = 4–6). Asterisks indicate mutants for which pCMBS inhibition was not determined due to low glucose transport activities.