Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B.
Two genes encoding distinct glutamate carrier proteins of Escherichia coli B were cloned into an E. coli K-12 strain by using a cosmid vector, pHC79. One of them was the gltS gene coding for a glutamate carrier of an Na+-dependent, binding protein-independent, and glutamate-specific transport system. The content of the glutamate carrier was amplified about 25-fold in the cytoplasmic membranes from a gltS-amplified strain. The gltS gene was located in a 3.2-kilobase EcoRI-MluI fragment, and the gene product was identified as a membrane protein with an apparent Mr of 35,000 in a minicell system. A gene designated gltP was also cloned. The transport activity of the gltP system in cytoplasmic membrane vesicles from a gltP-amplified strain was driven by respiratory substrates and was independent of the concentrations of Na+, K+, and Li+. An uncoupler, carbonylcyanide m-chlorophenylhydrazone, completely inhibited the transport activities of both systems, whereas an ionophore, monensin, inhibited only that of the gltS system. The Kt value for glutamate was 11 microM in the gltP system and 3.5 microM in the gltS system. L-Aspartate inhibited the glutamate transport of the gltP system but not that of the gltS system. Aspartate was taken up actively by membrane vesicles from the gltP-amplified strain, although no aspartate uptake activity was detected in membrane vesicles from a wild-type E. coli strain. These results suggest that gltP is a structural gene for a carrier protein of an Na+-independent, binding protein-independent glutamate-aspartate transport system.
Deguchi
Y
Y
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Yamato
I
I
Anraku
Y
Y
D015139:Blotting, Southern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D005838:Genotype; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000235:genetics*
D005971:Glutamates; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter