Transport of proteins into mitochondria

Translocational intermediates of precursor proteins of ATPase F1β subunit and cytochrome c1 across mitochondrial membranes were analyzed using two different approaches, transport at low temperature and transport after binding of precursor proteins to antibodies. Under both conditions precursors were partially transported into mitochondria in an energy-dependent manner. They were processed by the metalloprotease in the matrix but a major proportion of the polypeptide chains was still present at the outer face of the outer mitochondrial membrane. We conclude that transfer of precursors into the inner membrane or matrix space occurs through “translocation contact sites”; precursor polypeptides to F1β and cytochrome c1 enter the matrix space with the amino terminus first; and a membrane potential is required for the transmembrane movement on an amino-terminal “domain-like” structure but not for completing translocation of the major part of the polypeptides

Transport of ADP/ATP carrier into mitochondria

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

Proteinaceous receptors for the import of mitochondrial precursor proteins

Mild trypsin treatment of isolated Neurospora mitochondria strongly inhibits their ability to bind and import the precursors of several mitochondrial proteins. Evidence is presented for two proteins, the ADP/ATP carrier and the mitochondrial porin, that specific binding of the precursors to the outer surface of the mitochondria is affected by the protease treatment. We suggest that the receptors that mediate the import of these two precursors are proteinaceous. Treatment of mitochondria with elastase also inhibits the binding and import of the ADP/ATP carrier and the porin. In contrast the import of the precursors of subunits 2 and 9 of the mitochondrial proton-translocating ATPase was unaffected by elastase treatment at the concentrations used. We suggest that the import pathways of the latter two proteins are distinct from those of the ADP/ATP carrier and the porin

Transfer of proteins into mitochondria

The precursor form of Neurospora crassa mitochondrial ADP/ATP carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. If the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. Upon reestablishment of the membrane potential, the bound precursor is imported. This occurs without dissociation of the bound precursor from the mitochondrial surface. We conclude that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway

Protein folding causes an arrest of preprotein translocation into mitochondria in vivo

With vital yeast cells, a hybrid protein consisting of the amino- terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria. Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain. Thereby, three salient features of mitochondrial protein uptake in vivo were demonstrated: its posttranslational character; the requirement for unfolding of precursors; and import through translocation contact sites. The permanent occupation of translocation sites by the fusion protein inhibited the import of other precursors; it did, however, not lead to leakage of mitochondrial ions, implying the existence of a channel that is sealed around the membrane spanning polypeptide segment