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

The carboxyl-terminal membrane-spanning segment 8 of the glutamate transporter GltT of Bacillus stearother-mophilus was studied by cysteine-scanning mutagene-sis. 21 single cysteine mutants were constructed in a stretch ranging from Gly-374 to Gln-404. Two mutants were not expressed, four were inactive, and two showed severely reduced glutamate transport activity. Cysteine mutations at the other positions were well tolerated. Only the two most amino- and carboxyl-terminal mu-tants (G374C, I375C, S399C, and Q404C) could be labeled with the large thiol reagent fluorescein maleimide, indi-cating unrestricted access and a location in a loop struc-ture outside the membrane. The labeling pattern of these mutants using membrane- permeable and-imper-meable thiol reagents showed that the N and C termin