The Translocase of the Outer Membrane of Mitochondria (TOM complex)

The TOM complex, a multisubunit assembly in the mitochondrial outer
membrane, mediates targeting and membrane translocation of
virtually all nuclear-encoded mitochondrial preproteins analyzed so
far. In the present study the mechanisms by which the TOM complex
recognizes different precursor proteins and translocates them
across the outer membrane were investigated. In a first part of
study the isolated TOM complex was analyzed for its ability to
interact with preproteins with N-terminal targeting signals. The
TOM translocase was found to bind precursor proteins efficiently in
a specific manner in the absence of chaperones and lipids in a
bilayer structure. Following the initial binding, the presequence
was transferred into the translocation pore in a step that required
unfolding of the mature part of the preprotein. This translocation
step was mediated also by protease-treated TOM holo complex that
contains almost exclusively Tom40. The TOM core complex consisting
of Tom40, Tom22, Tom5, Tom6 and Tom7 represents a molecular machine
that can recognize and partially translocate mitochondrial
precursor proteins. In a second part of study the interaction of
BCS1 precursor with the TOM complex was investigated. BCS1 belongs
to the group of proteins with internal, non-cleavable import
signals. The information for import and intramitochondrial sorting
of BCS1 was localized to the region consisting of amino acid
residues 1-126. Three sequence elements were identified in this
region: (i) a transmembrane domain (amino acid residues 45-68),
(ii) a presequence-like helix (residues 69-83), and (iii) an
import-auxiliary sequence (residues 84-126). The contribution of
each of these elements to import was studied. The transmembrane
domain was found not to be required for stable binding to the TOM
complex. The Tom receptors (Tom70, Tom22 and Tom20), as determined
by peptide scan analysis, had no affinity for peptides
corresponding to the transmembrane domain. They did interact with
the presequence-like helix, yet the highest binding was to the
region covering residues 92-126. This latter region represents a
novel type of signal with targeting and sorting function. It is
recognized by all three known mitochondrial import receptors
demonstrating their capacity to decode various targeting signals.
The results of the present study suggest that the BCS1 precursor
crosses the TOM complex as a loop structure. This is in contrast to
preproteins with cleavable presequences which enter the TOM complex
in a linear fashion with the N-terminal first. Once the precursor
emerges from the TOM complex, all three structural elements are
essential for the intramitochondrial sorting to the inner membrane.