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 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 alpha-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 alpha-helix. The hydrophilic face of the helix lines an aqueous pore and contains many residues that are important for activity.
Slotboom
D J
DJ
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands.
Konings
W N
WN
Lolkema
J S
JS
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D027322:Amino Acid Transport System X-AG; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D003545:Cysteine; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D001426:Bacterial Proteins; D003545:Cysteine