Characterization of Translocation Contact Sites Involved in the Import of Mitochondrial Proteins

Import of proteins into the mitochondrial matrix requires
translocation across two membranes. Translocational intermediates
of mitochondrial proteins, which span the outer and inner membrane
simultaneously and thus suggest that translocation occurs in one
step, have recently been described (Schleyer, M., and W. Neupert,
1985, Cell, 43:339-350). In this study we present evidence that
distinct membrane areas are involved in the translocation process.
Mitochondria that had lost most of their outer membrane by
digitonin treatment (mitoplasts) still had the ability to import
proteins. Import depended on proteinaceous structures of the
residual outer membrane and on a factor that is located between the
outer and inner membranes and that could be extracted with
detergent plus salt. Translocational intermediates, which had been
preformed before fractionation, remained with the mitoplasts under
conditions where most of the outer membrane was subsequently
removed. Submitochondrial vesicles were isolated in which
translocational intermediates were enriched. Immunocytochemical
studies also suggested that the translocational intermediates are
located in areas where outer and inner membranes are in close
proximity. We conclude that the membrane-potential-dependent import
of precursor proteins involves translocation contact sites where
the two membranes are closely apposed and are linked in a stable
manner.