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.