Among four
proteins that interacted specifically with A-RAF
protein was human protein that has 74% homology to yeast TIM44
protein. We named it hTIM44.
yTIM44 was shown to function in the protein import from cytosole into mitochondria.
It is located in the inner mitochondrial membrane and involved in translocation
of polypeptides across it. The second A-RAF interacting protein contained
22 TPR repeats. Yeast outer-membrane mitochondrial import receptor
TOM70
also has multiple
TPR
repeats, therefore we called it hTOM.
Thus, these
two interactions suggested that A-RAF could be imported into mammalian
mitochondria. To prove this hypothesis, I have isolated
mitochondria from rat liver and showed the A-RAF presence in it by
Western blot analysis with A-RAF specific antibodies from Santa-Cruz
Biotechnology. I also showed that A-RAF was protected from proteolysis
in both intact and osmotically treated mitochondria, suggesting that A-RAF
is located in both mitochondrial matrix and in inter-membrane space.
To prove further that A-RAF is present in mitochondria I have performed
immunogold labeling of purified rat liver mitochondria with A-RAF specific
antibodies. 95% of the gold label was inside mitochondria in
both inter-membrane space and matrix.