Mitochondrial precursor proteins are imported through a hydrophilic membrane environment

We have analyzed how translocation intermediates of imported
mitochondrial precursor proteins, which span contact sites,
interact with the mitochondrial membranes. F1-ATPase subunit β(F1β)
was trapped at contact sites by importing it into Neurospora
mitochondria in the presence of low levels of nucleoside
triphosphates. This F1β translocation intermediate could be
extracted from the membranes by treatment with protein denaturants
such as alkaline pH or urea. By performing import at low
temperatures, the ADP/ATP carrier was accumulated in contact sites
of Neurospora mitochondria and cytochrome b2 in contact sites of
yeast mitochondria. These translocation intermediates were also
extractable from the membranes at alkaline pH. Thus, translocation
of precursor proteins across mitochondrial membranes seems to occur
through an environment which is accessible to aqueous perturbants.
We propose that proteinaceous structures are essential components
of a translocation apparatus present in contact sites.