Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria.
The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been expressed at an abundant level in Escherichia coli. It accumulates in the bacterium as inclusion bodies, and none of the protein was detected in the bacterial inner membrane. The mitochondrial ADP/ATP carrier, a member of the same super-family of transport proteins as the oxoglutarate carrier, has also been expressed in E. coli. However, the expression of the ADP/ATP carrier in bacteria retards their growth, and so the levels of expression that were attained were lower than those of the oxoglutarate carrier. The oxoglutarate carrier inclusion bodies have been disaggregated with the detergent N-dodecanoyl-sarcosine, and the protein has been incorporated into liposomes. In its ability to transport oxoglutarate and malate and other known substrates of the carrier in mitochondria, and in its inhibition characteristics by a wide range of non-competitive and competitive inhibitors, this reconstituted oxoglutarate carrier is similar to the natural protein in the inner membranes of mitochondria, and to the carrier that has been purified from mitochondria and reconstituted in liposomes. These experiments remove significant obstacles to crystallization trials and to site-directed mutagenesis of the oxoglutarate carrier.
Fiermonte
G
G
Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.
Walker
J E
JE
Palmieri
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli; D007656:Ketoglutaric Acids; D007700:Kinetics; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data
D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; C021723:oxoglutarate translocator; D000226:Mitochondrial ADP, ATP Translocases