Cysteine-scanning Mutagenesis Reveals a Highly Amphipathic, Pore-lining Membrane-spanning Helix in the Glutamate Transporter GltT

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands.
Journal of Biological Chemistry (Impact Factor: 4.6). 05/2001; DOI: 10.1074/jbc.M011064200
Source: OAI

ABSTRACT The carboxyl-terminal membrane-spanning segment 8 of the glutamate transporter GltT of Bacillus stearothermophilus was studied by cysteine-scanning mutagenesis. 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 mutants (G374C, I375C, S399C, and Q404C) could be labeled with the large thiol reagent fluorescein maleimide, indicating unrestricted access and a location in a loop structure outside the membrane. The labeling pattern of these mutants using membrane- permeable and -impermeable thiol reagents showed that the N and C termini of the mutated stretch are located extra- and intracellularly, respectively. Thus, the location of the membrane-spanning segment was confined to a stretch of 23 residues between 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 small and flexible reagent 2-aminoethyl methanethiosulfonate hydrobromide only, suggesting accessibility via a narrow aqueous pore. When the region was modeled as an α-helix, all positions at which cysteine mutations lead to inactive or severely impaired transporters cluster on one face of this helix. The inactive mutants showed neither proton motive force-driven uptake activity nor exchange activity nor glutamate binding. The results indicate that transmembrane segment 8 forms an amphipathic α-helix. The hydrophilic face of the helix lines an aqueous pore and contains many residues that are important for activity.

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