Protein import into mitochondria requires the energy of ATP hydrolysis inside and/or outside mitochondria. Although the role of ATP in the mitochondrial matrix in mitochondrial protein import has been extensively studied, the role of ATP outside mitochondria (external ATP) remains only poorly characterized. Here we developed a protocol for depletion of external ATP without significantly reducing the import competence of precursor proteins synthesized in vitro with reticulocyte lysate. We tested the effects of external ATP on the import of various precursor proteins into isolated yeast mitochondria. We found that external ATP is required for maintenance of the import competence of mitochondrial precursor proteins but that, once they bind to mitochondria, the subsequent translocation of presequencecontaining proteins, but not the ADP/ATP carrier, proceeds independently of external ATP. Because depletion of cytosolic Hsp70 led to a decrease in the import competence of mitochondrial precursor proteins, external ATP is likely utilized by cytosolic Hsp70. In contrast, the ADP/ATP carrier requires external ATP for efficient import into mitochondria even after binding to mitochondria, a situation that is only partly attributed to cytosolic Hsp70.Many eukaryotic proteins have to move across one or more biological membrane(s) to reach their destinations after synthesis (1). Protein translocation across the organellar membranes is mediated by translocators or translocases, which are membrane-protein complexes and provide a protein-conducting channel through which organellar proteins thread, in most cases, in unfolded conformations. Several translocation systems can actively unfold precursor proteins and achieve their vectorial movement across the membranes by using various forms of energy (2, 3).Mitochondria consist of the outer and inner membranes and two aqueous compartments, the intermembrane space and matrix. The vast majority of mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. Precursors of most matrix proteins and many inner membrane proteins have a positively charged presequence that contains targeting information for mitochondria and is removed by the processing peptidase in the matrix. These proteins utilize the translocase of the outer mitochondrial membrane (TOM) 1 to cross the outer membrane and the translocase of the inner mitochondrial membrane, the TIM23 complex, for translocation across the inner membrane (4, 5). The targeting information in the presequence is recognized by Tom20, a general presequence receptor subunit of the TOM complex (6). Polytopic inner membrane proteins, including members of the carrier protein family such as the ADP/ATP carrier (AAC), are not synthesized with cleavable presequences but instead contain internal mitochondrial targeting signals. Members of the carrier protein family utilize the TOM complex to cross the outer membrane and another translocase of the inner membrane, the TIM22 complex, for integration into the inner membrane (7,8). These...

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