Protein sorting to mitochondria

According to the endosymbiont hypothesis, mitochondria have lost the autonomy of their prokaryotic ancestors. They have to import most of their proteins from the cytosol because the mitochondrial genome codes for only a small percentage of the polypeptides that reside in the organelle. Recent findings show that the sorting of proteins into the mitochondrial subcompartments and their folding and assembly follow principles already developed in prokaryotes. The components involved may have structural and functional equivalents in bacteria

Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis

Mitochondrial heat-shock protein hsp60 functions in the folding of proteins imported into mitochondria. Folding occurs at the surface of hsp60 in an ATP-mediated reaction, followed by release of the bound polypeptides. We propose that hsp60 catalyses protein folding

Polypeptides traverse the mitochondrial envelope in an extended state

Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through the contact sites between outer and inner mitochondrial membranes. The molecular mechanism of membrane translocation of precursor proteins is largely unclear. For this report, various hybrid proteins between portions of the precursor of cytochrome b2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites. We unexpectedly found that about 30 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes. This suggests linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes

Translocation arrest by reversible folding of a precursor protein imported into mitochondria

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number