Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system.
The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.
Hoshino
T
T
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Kose-Terai
K
K
Sato
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000597:Amino Acids, Branched-Chain / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli; D007930:Leucine / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D011510:Proteolipids; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D012995:Solubility; D014764:Viral Proteins
D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D008565:Membrane Proteins; D011510:Proteolipids; D014764:Viral Proteins; C020485:proteoliposomes; D000255:Adenosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D007930:Leucine