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Eur J Biochem. 2002 Jul;269(13):3172-81.

Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids.

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1
Pflanzenphysiologie, Universität Kaiserslautern, Erwin-Schrödinger-Strasse, D-67663 Kaiserslautern, Germany.

Abstract

The expression of mitochondrial and hydrogenosomal ADP/ATP carriers (AACs) from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 in Escherichia coli allows a functional integration of the recombinant proteins into the bacterial cytoplasmic membrane. For AAC1 and AAC2 from rat, apparent Km values of about 40 microm for ADP, and 105 microm or 140 microm, respectively, for ATP have been determined, similar to the data reported for isolated rat mitochondria. The apparent Km for ATP decreased up to 10-fold in the presence of the protonophore m-chlorocarbonylcyanide phenylhydrazone (CCCP). The hydrogenosomal AAC isolated from the chytrid fungus Neocallimastix spec. L2 exhibited the same characteristics, but the affinities for ADP (165 microm) and ATP (2.33 mm) were significantly lower. Notably, AAC1-3 from Arabidopsis thaliana and AAC1 from Solanum tuberosum (potato) showed significantly higher external affinities for both nucleotides (10-22 microm); they were only slightly influenced by CCCP. Studies on intact plant mitochondria confirmed these observations. Back exchange experiments with preloaded E. coli cells expressing AACs indicate a preferential export of ATP for all AACs tested. This is the first report of a functional integration of proteins belonging to the mitochondrial carrier family (MCF) into a bacterial cytoplasmic membrane. The technique described here provides a relatively simple and highly reproducible method for functional studies of individual mitochondrial-type carrier proteins from organisms that do not allow the application of sophisticated genetic techniques.

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