Cysteine-scanning mutagenesis reveals a highly amphipathic, pore-lining membrane-spanning helix in the glutamate transporter GltT

The carboxyl terminal membrane-spanning segment 8 of the glutamate transpor ter GltT of Bacillus stearothermophilus was studied by cysteine-scanning mu tagenesis. 21 single cysteine mutants were constructed in a stretch ranging from Gly-574 to Gln-404, Two mutants were not expressed, four were inactiv e, and two showed severely reduced glutamate transport activity. Cysteine m utations at the other positions were well tolerated. Only the two most amin o- and carboxyl-terminal mutants (G374C, I375C, S399C, and Q404C) could be labeled with the large thiol reagent fluorescein maleimide, indicating unre stricted access and a location in a loop structure outside the membrane. Th e labeling pattern of these mutants using membrane- permeable and -impermea ble thiol reagents showed that the N and C termini of the mutated stretch a re located extra- and intracellularly, respectively. Thus, the location of the membrane-spanning segment was confined to a stretch of 23 residues betw een Gly-374 and Ser-399. Cysteine residues in three mutants in the central part of the segment (M381C, V388C, and N391C) could be labeled with the sma ll and flexible reagent a-aminoethyl methanethiosulfonate hydrobromide only , suggesting accessibility via a narrow aqueous pore. When the region was m odeled as an cu-helix, all positions at which cysteine mutations lead to in active or severely impaired transporters cluster on one face of this helix, The inactive mutants showed neither proton motive force-driven uptake acti vity nor exchange activity nor glutamate binding. The results indicate that transmembrane segment 8 forms an amphipathic alpha -helix, The hydrophilic face of the helix lines an aqueous pore and contains many residues that ar e important for activity.