ADP‐ATP Carrier of Saccharomyces cerevisiae contains a mitochondrial import signal between amino acids 72 and 111

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Abstract
The ADP‐ATP carrier (also referred to as the adenine nucleotide translocator) of Saccharomyces cerevisiae is encoded by a nuclear gene, translated in the cytosol, and imported into the mitochondrial inner membrane. In order to study the determinants of mitochondrial import, a series of fusion proteins, consisting of the first 21, 72, and 111 amino acids of the ADP‐ATP carrier, joined to mouse dihydrofolate reductase were generated. Dihydrofolate reductase is a cytosolic protein that does not bind mitochondria. The reticulocyte lysate reaction containing the 35S‐methionine‐labeled protein was incubated with mitochondria in a buffer containing 3% BSA. Following incubation for import, the reactions were treated with 1 mM PMSF or 25 μg/ml proteinase K; mitochondria were reisolated and analyzed by gel electrophoresis. The 21 and 72 amino acid hybrid proteins showed a low level of binding to mitochondria: the bound form was entirely protease accessible. The 111 amino acid hybrid protein was imported to a protease‐protected location within mitochondria. It is concluded that the first 72 amino acids of the ADP‐ATP carrier do not suffice to import the protein into mitochondria and that the region between amino acids 72 and 111, a region that contains a transmembrane‐spanning domain, constitutes at least part of the mitochondrial import signal.