Isolation and characterisation of the intermembrane space components of the mitochondrial TIM22 protein import machinery of Neurospora crassa

Mitochondria are essential cellular organelles of eukaryotic
organisms, which import most of their proteinaceous constituents
from the cytoplasm. Two mitochondrial membranes contain different
translocation machineries which are involved in the import and
proper sorting of mitochondrial precursor proteins. The TIM22
translocase in the inner mitochondrial membrane mediates the import
of polytopic proteins into this membrane. In addition to the
membrane integrated components Tim22 and Tim54, the TIM22
translocase possesses components in the intermembrane space, termed
Tim9 and Tim10. In the present study, the tim9 and tim10 genes of
the TIM22 translocase of N. crassa were identified. The structural
and functional characteristics of the corresponding gene products,
the Tim9 and Tim10 proteins, were examined. Tim9 was demonstrated
to be an essential protein. The Tim9 and Tim10 proteins were shown
to build a 70-80 kDa heterohexameric complex in the mitochondrial
intermembrane space. The isolated Tim9•Tim10 complex had the same
oligomeric structure as the native one, and it proved fully
functional in interacting in vitro with its physiological
substrate, the ADP/ATP carrier (AAC). Peptide library screens were
performed to determine the structural determinants of the
substrates that are recognised by the Tim9•Tim10 complex. Efficient
binding to the regions covering residues of the hydrophobic
membrane spanning domains and of the connecting hydrophilic loops
was observed. In this way, Tim9 and Tim10 proteins interact with
their substrates, while the hydrophobic regions of the substrates
are still present in the TOM complex and thereby protected from the
aqueous environment of the intermembrane space compartment.
Furthermore, when enclosed into proteoliposomes containing the
reconstituted TOM complex, Tim9•Tim10 complex specifically promoted
the translocation of the AAC precursor. Hence, the Tim9•Tim10
complex and the TOM complex are both necessary and sufficient to
facilitate translocation of carrier proteins across the outer
mitochondrial membrane. Finally, peptide screens and chemical
cross-linking experiments were used to identify the precursor of N.
crassa Tim23 protein as a novel substrate of the Tim9•Tim10
complex.