Precursor to ADP/ATP carrier synthesized in vitro is transferred into isolated mitochondria to a protease-resistant location. This process requires an electrical potential across the inner membrane. We show now that precursor imported in a cell-free system exhibits the same resistance to protease as the mature endogenous carrier. Furthermore, transferred protein, but not receptor-associated precursor, binds carboxy-atractyloside, a specific inhibitor of the mature carrier and can be isolated by the purification procedure for the mature carrier. At least 70% of the precursor associated with mitochondria in the presence of a membrane potential acquires this functional characteristic. Finally, the binding of carboxyatractyloside can be modulated by treatment of the imported protein with sulfhydryl reagents in a manner indistinguishable from the authentic carrier protein. We conclude that import in vitro leads to correct assembly and orientation of the ADP/ATP carrier in the mitochondria
