Among four
proteins that interacted specifically with A-RAF was novel human
protein that had 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 specific interacting protein
contains 22 TPR repeats. Yeast outer-membrane mitochondrial import
receptor TOM70 also has multiple
TPR repeats, therefore we
call our novel protein hTOM.
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 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 was located in both mitochondrial matrix and in inter-membrane
space. To prove further that A-RAF was 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.
More details are available from full text on-line:
Yuryev A, Ono M, Goff SA,
Macaluso F, Wennogle LP.
Isoform-Specific Localization
of A-RAF in Mitochondria.
Mol Cell Biol. 2000 Jul 1; 20(13): 4870-4878. PMID: 10848612