Solute carrier 11 cation symport requires distinct residues in transmembrane helices 1 and 6.
Ubiquitous solute carriers 11 (SLC11) contribute to metal-ion homeostasis by importing Me(2+) and H(+) into the cytoplasm. To identify residues mediating cation symport, Escherichia coli proton-dependent manganese transporter (MntH) was mutated at five SLC11-specific transmembrane (TM) sites; each mutant activity was compared with wild-type MntH, and the biochemical results were tested by homology threading. Cd(2+) and H(+) uptake kinetics were analyzed in whole cells as a function of pH and temperature, and right-side out membrane vesicles were used to detail energy requirements and to probe site accessibility by Cys replacement and thiol modification. This approach revealed that TM segment 1 (TMS1) residue Asp(34) couples H(+) and Me(2+) symport and contributes to MntH forward transport electrogenicity, whereas the TMS6 His(211) residue mediates pH-dependent Me(2+) uptake; MntH Asn(37), Asn(250), and Asn(401) in TMS1, TMS7, and TMS11 participate in transporter cycling and/or helix packing interactions. These biochemical results fit the LeuT/SLC6 structural fold, which suggests that conserved peptide motifs Asp(34)-Pro-Gly (TMS1) and Met-Pro-His(211) (TMS6) form antiparallel "TM helix/extended peptide" boundaries, lining a "pore" cavity and enabling H(+)-dependent Me(2+) import.
Courville
Pascal
P
INRS-Institut Armand-Frappier, 531 Boulevard des prairies, Laval, Québec, Canada.
Urbankova
Eva
E
Rensing
Christopher
C
Chaloupka
Roman
R
Quick
Matthias
M
Cellier
Mathieu F M
MF
D020816:Amino Acid Motifs / Q000502:physiology; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D002104:Cadmium / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology; D007700:Kinetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D011522:Protons; D027981:Symporters / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; C492412:MntH protein, E coli; D011522:Protons; D027981:Symporters; D002104:Cadmium