A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop.
Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft. The proteins belong to a large family of secondary transporters, which includes bacterial glutamate transporters. The C-terminal half of the glutamate transporters is well conserved and thought to contain the translocation path and the binding sites for substrate and coupling ions. A serine-rich sequence motif in this part of the proteins is located in a putative intracellular loop. Cysteine-scanning mutagenesis was applied to this loop in the glutamate transporter GltT of Bacillus stearothermophilus. The loop was found to be largely intracellular, but three consecutive positions in the conserved serine-rich motif (S269, S270, and E271) are accessible from both sides of the membrane. Single-cysteine mutants in the serine-rich motif were still capable of glutamate transport, but modification with N-ethylmaleimide blocked the transport activity in six mutants (T267C, A268C, S269C, S270C, E271C, and T272C). Two milimolars L-glutamate effectively protected against the modification of the cysteines at position 269-271 from the periplasmic side of the membrane but was unable to protect cysteine modification from the cytoplasmic side of the membrane. The results indicate that the conserved serine-rich motif in the glutamate transporter forms a reentrant loop, a structure that is found in several ion channels but is unusual for transporter proteins. The reentrant loop is of crucial importance for the function of the glutamate transporter.
Slotboom
D J
DJ
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands.
Sobczak
I
I
Konings
W N
WN
Lolkema
J S
JS
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000502:physiology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D012694:Serine
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D018698:Glutamic Acid; D012694:Serine; D003545:Cysteine; D005033:Ethylmaleimide