Transport of Proteins into Mitochondria. Posttranslational Transfer of ADP/ATP Carrier into Mitochondria in vitro

Abstract
The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner membrane and it is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is transferred into mitochondria in a posttranslational manner. Evidence for such a mechanism and details are provided here. In homologous and heterologous translation systems the newly synthesized ADP/ATP carrier protein is present in the postribosomal supernatant. Analysis by density gradient centrifugation and gel filtration shows that the ADP/ATP carrier molecules in the postribosomal fraction are present as soluble complexes with apparent MW of .apprx. 120,000 and .apprx. 500,000 or larger. The carrier binds detergents such as Triton X-100 and deoxycholate forming mixed micelles with MW of .apprx. 200,000-.apprx. 400,000. Incubation of a postribosomal supernatant of a reticulocyte lysate containing newly synthesized ADP/ATP carrier with mitochondria isolated from Neurospora spheroplasts results in efficient transfer of the carrier into mitochondria. About 20-30% of the transferred carrier are resistant to proteinase in whole mitochondria. The authentic mature protein is also largely resistant to proteinase in whole mitochondria and sensitive after lysing of mitochondria with detergent. Integrity of mitochondria is a prerequisite for translocation into proteinase resistant position. The transfer in vitro into a proteinase-resistant form is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding. The posttranslational transfer of ADP/ATP carrier occurs via the cytosolic space through a soluble oligomeric precursor form. This precusor is taken up by intact mitochondria into an integral position in the membrane. These findings are considered to be of general importance for the intracellular transfer of insoluble membrane proteins. Such proteins apparently can exist in a water-soluble form as precursors and upon integration into the membrane undergo a conformational change. Uptake into the membrane may involve the cleavage of an additional sequence in some proteins, but this appears not to be a prerequisite as demonstrated by the ADP/ATP carrier protein.