Import of Proteins into Mammalian Mitochondria

Abstract

<p>Most mitochondrial proteins are encoded in the nuclear genome and imported into mitochondria after their synthesis on cytoplasmic ribosomes. Two rat liver mitochondrial proteins, malate dehydrogenase and the adenine nucleotide carrier, were selected to study the mechanism of importing nuclear coded mitochondrial proteins into the organelle.</p> <p>Rat liver proteins were synthesized in vitro and the nascent forms of the two proteins were immunoprecipitated by monospecific antibodies against each protein. Size determination by sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that each precursor protein is about 1,500-2,000 daltons larger than its mature monomeric form. In contrast to secretory proteins, both proteins are exclusively synthesized on membrane free polysomes. Molecular mass sieving experiments indicated that both precursor proteins exist as aggregates or complexes after their synthesis. These aggregates or complexes may play a role in transfering the two precursor proteins to the mitochondrial outer membrane.</p> <p>The import of both precursor proteins into mitochondria was studied with an in vitro system, consisting of the translation mixture and mitochondria isolated from Chinese hamster ovary cells. The precursor of malate dehydrogenase, synthesized in vitro, could be imported into isolated mitochondria in the absence of protein synthesis. The import was accompanied with the conversion of the larger precursor to the mature monomeric size. The newly processed precursor, in mitochondria, was insensitive to externally added protease, suggesting that the protein had been translocated into at least the inter-membrane space, if not the matrix. The proteolytic cleavage was arrested by the chelator, o-phenanthroline, indicating the protease responsible for the conversion requires metallic cations as a cofactor. Time course studies showed that import and processing of the precursor was completed within 10 to 30 min at 30°C and the precursor was converted to the mature size by a single cleavage. Import was found to depend on the electrochemical potential across the inner membrane, as shown by the inhibitory effect of the uncoupler, carbonyl cyanide m-chlorophenylhydrazone.</p> <p>The same in vitro import system was used to investigate the import of the precursor of the rat liver adenine nucleotide carrier. Although processing of the precursor to its mature size was not observed, there were indications that the precursor was imported into mitochondria: (1) a time-dependent transfer of the precursor from the incubation solution to mitochondria was observed, and the process was greatly reduced in presence of an uncoupler. (2) Unlike the precursor in the incubation solution, the precursor immunoprecipitated from the mitochondrial fraction was protease insensitive. The reasons for the lack of processing of the imported precursor in the in vitro system is not known. However, the results suggest that proteolytic processing of the precursor is not an obligatory step in its import.</p> <p>The success in establishing an in vitro system in which the processing of the precursor of rat liver malate dehydrogenase occurred, allowed probing of the details of the import process. In the presence of an uncoupler, binding, but not processing, of the precursor to mitochondria was observed. Further experiments showed that the binding process fulfils most of the criteria for a genuine ligand-receptor interaction. It is temperature insensitive and completed very rapidly at 0°C. The binding process is not inhibited by the presence of an uncoupler. The bound precursor is protease sensitive, indicating that its putative receptor is located on the mitochondrial outer membrane. An apparent saturation of the binding sites was observed with a fixed amount of mitochondria and excess amount of in vitro synthesized proteins. Most importantly, it was observed that processing of bound precursor was independent of the volume of reaction mixture, suggesting that bound precursor was directly processed to its mature size. In contrast, processing of unbound precursor was dependent on reaction volume. These results suggest that binding of the precursor of malate dehydrogenase to the mitochondrial outer membrane is an intermediate step in the import process.</p> <p>Mitochondrial malate dehydrogenase is a NAD⁺-dependent dehydrogenase. The enzyme has been purified to homogeneity from different sources by the affinity column, 5-AMP-Sepharose. However, the precursor of rat liver malate dehydrogenase synthesized in vitro did not bind to the affinity column, indicating the absence of a NAD⁺-binding site on the precursor protein. After conversion to the mature size by isolated mitochondria, the processed protein bound to the affinity column and was eluted with the endogenous enzyme by 40 uM NADH. These results strongly indicate that processing of the precursor to its mature size is followed by conformational changes resulting in the formation of a functional NAD⁺-binding site. This conclusion is supported by studies with antisera prepared against the sodium dodecyl sulphate denatured enzyme, which are conformationally specific. The antiserum recognized only the sodium dodecyl sulphate denatured mature protein and the in vitro processed precursor protein, but not the native functional enzyme. However, the antiserum could immunoprecipitate the precursor protein with or without prior denaturation. The above results suggest that the in vitro imported precursor protein has been converted into a functional enzyme, and indicate that the in vitro import system reflects the process that occurs in vivo.</p>