Membrane topology of the C-terminal half of the neuronal, glial, and bacterial glutamate transporter family.
Secondary glutamate transporters in neuronal and glial cells in the mammalian central nervous system remove the excitatory neurotransmitter glutamate from the synaptic cleft and prevent the extracellular glutamate concentration to rise above neurotoxic levels. Secondary structure prediction algorithms predict 6 transmembrane helices in the first half of the transporters but fail in the C-terminal half where no clear helix-loop-helix motif is resolved in the hydropathy profile of the primary sequences. A number of previous studies have emphasized the importance of the C-terminal half of the molecules for the function. Here we determine the membrane topology of the C-terminal half of the glutamate transporters by applying the phoA gene fusion technique to the homologous bacterial glutamate transporter of Bacillus stearothermophilus. High sequence conservation and very similar hydropathy profiles in the C-terminal half warrant a similar folding as in the glutamate transporters of the mammalian central nervous system. The C-terminal half contains four putative transmembrane helices. The strong hydrophobic moment and substitution moment of the most C-terminal helix X that point to opposite faces of the helix suggest that the helix faces the lipid environment with its least conserved, hydrophobic face and the interior of the protein with its well conserved, hydrophilic face. Residues that were shown before to be critical for function cluster in helix X and VII.
Slotboom
D J
DJ
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. j.s.lolkema@biol.rug.nl
Lolkema
J S
JS
Konings
W N
WN
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D015153:Blotting, Western; D001411:Geobacillus stearothermophilus; D008969:Molecular Sequence Data; D009457:Neuroglia / Q000737:chemistry*; D009474:Neurons / Q000737:chemistry*
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D000469:Alkaline Phosphatase