Previously, the role of residues in the ADP/ATP carrier (AAC) from Saccharomyces cerevisiae has been studied by mutagenesis, but the dependence of mitochondrial biogenesis on functional AAC impedes segregation of the mutational effects on transport and biogenesis. Unlike other mitochondrial carriers, expression of the AAC from yeast or mammalians in Escherichia coli encountered difficulties because of disparate codon usage. Here we introduce the AAC from Neurospora crassa in E. coli, where it

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... accumulated in inclusion bodies and establish the reconstitution conditions. AAC expressed with heat shock vector gave higher activity than with pET-3a. Transport activity was absolutely dependent on cardiolipin. The 10 single mutations of intrahelical positive residues and of the matrix repeat (؉X؉) motif resulted in lower activity, except of R245A. R143A had decreased sensitivity toward carboxyatractylate. The ATP-linked exchange is generally more affected than ADP exchange. This reflects a charge network that propagates positive charge defects to ATP 4؊ more strongly than to ADP 3؊ transport. Comparison to the homologous mutants of yeast AAC2 permits attribution of the roles of these residues more to ADP/ATP transport or to AAC import into mitochondria. The ADP/ATP carrier (AAC) 1 is involved in the last step of the oxidative phosphorylation system by delivering ATP into the cytosol. Its slow intrinsic turnover is a result of unusually large and highly charged substrates; it is the most strongly expressed member of the mitochondrial carrier family (1). The AAC has been instrumental in understanding certain principles of transport mechanism, such as the "single binding gated pore mechanism" (2-4) and the "induced transition fit" (5, 6) of transport catalysis. The drastic conformation changes linked to the transport are well documented (7, 8) . Based on its sequence