Biogenesis of proteins of the mitochondrial intermembrane space

All intermembrane space (IMS) proteins are synthesized in the
cytosol and have to be imported into mitochondria. Many proteins of
the IMS lack typical N-terminal targeting signals and are
characterized by a small molecular mass and highly conserved
cysteine residues present in characteristic patterns. These
proteins cross the outer membrane of mitochondria via the TOM
complex and need their cysteine residues for the efficient
retention in the IMS. The aim of this study was to analyse whether
specific factors are required for the import of these proteins into
the mitochondrial IMS. The candidate protein, later termed Mia40
(mitochondrial intermembrane space import and assembly), was
structurally and functionally characterized. The experiments
presented here confirmed the mitochondrial location of Mia40 and
determined its topology. Mia40 contains a classical N-terminal
mitochondrial targeting signal followed by a hydrophobic segment.
It is anchored in the inner membrane by a hydrophobic stretch and
exposes a large C-terminal domain to the IMS. This domain harbours
six highly conserved cysteine residues forming a CXC-CX9C-CX9C-
motif (X represents non-cysteine amino acid residues). Since Mia40
is essential for viability of yeast, a strain harbouring the MIA40
gene under control of the glucose-repressible GAL10 promoter was
used to study the function of Mia40 in mitochondria. Depletion of
Mia40 resulted in strongly reduced levels of Tim13, Cox17 and of
other IMS proteins with cysteine motifs, which was due to the
impairment of their import into mitochondria. Mia40 is directly
involved in the translocation of the small IMS proteins with
conserved cysteine motifs: the newly imported IMS proteins form
mixed disulfide intermediates with Mia40. In mitochondria, the
majority of Mia40 is present in the oxidized state, thus allowing
the formation of the mixed disulfide intermediates in an
isomerization reaction. Subsequently, Mia40 transfers the disulfide
bond from the mixed disulfide to the substrate proteins and thereby
triggers the folding and the trapping of these proteins in the IMS.
Mia40 is left in a partially reduced state and a reoxidation step
is required for the next round of import. Erv1 is an essential
FAD-containing sulfhydryl oxidase present in the IMS of fungi,
plants and animals. The import of Tim13 was less efficient in
mitochondria depleted of Erv1 and Mia40 interacted directly with
Erv1 via disulfide bonds. In addition, the depletion of Erv1
affected the redox state of Mia40, which accumulated in a partially
reduced state, suggesting that Erv1 is required for the recovery of
the oxidized state of Mia40. Thus, Mia40 and Erv1 form a disulfide
relay system mediating the import of small cysteine-rich proteins
into the IMS. Erv1 passes its electrons further to cytochrome c,
linking the import of small IMS proteins to the respiratory chain
activity. Notably, Erv1 is not only a component but also a
substrate of the disulfide relay system. It represents a novel type
of substrate of the Mia40-mediated pathway. Thus, this pathway
appears to be quite versatile and not limited to proteins with twin
CX3C or twin CX9C motifs. The conserved cysteine residues in Mia40
are crucial for its function. Using single and double cysteine
mutants of Mia40, it was possible to assign specific roles to each
cysteine residue. In the oxidized state of Mia40 all cysteine
residues form intramolecular disulfide bonds. The first two
cysteine residues in the CPC motif compose a redox-sensitive
disulfide bridge and breaking of this disulfide leads to Mia40 in
the partially reduced state. The disulfide bond formed by the first
two cysteine residues in Mia40 seems to be involved in the
interaction with Erv1 and the substrate proteins, suggesting that
it is essential for the catalysis of redox reactions of Mia40. The
two other disulfide bonds connect the two CX9C fragments in Mia40
and most likely play a structural role. Taken together, the
essential protein Mia40 is the central component of a novel
translocation pathway. Mia40 together with Erv1 forms a disulfide
relay system required for the import of small cysteine-rich
proteins into the IMS of mitochondria.