Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters.
We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is approximately 30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form. Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 x 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.
Reig
Núria
N
Institute for Research in Biomedicine, Barcelona Science Park and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, E-08028 Barcelona, Spain.
del Rio
César
C
Casagrande
Fabio
F
Ratera
Mercè
M
Gelpí
Josep Lluís
JL
Torrents
David
D
Henderson
Peter J F
PJ
Xie
Hao
H
Baldwin
Stephen A
SA
Zorzano
Antonio
A
Fotiadis
Dimitrios
D
Palacín
Manuel
M
D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
D026921:Amino Acid Transport Systems, Neutral; D029968:Escherichia coli Proteins; D008081:Liposomes; D013912:Threonine; D012694:Serine