Biochemical characterization of Human Tim50, the major receptor of the mitochondrial protein import machinery.

The mitochondrion is an essential organelle that play key roles in hosting metabolic and bioenergetic processes and in modulation of life and death signals such as of apoptosis. Most of mitochondrial proteins are nuclear- encoded and translated in the cytosol. Subsequently, they are translocated into the mitochondrion via specialized translocation complexes. TIM23, one of the translocase complexes of the inner mitochondrial membrane, allows precursor proteins to pass through or into the inner membrane. Tim50 is a subunit of TIM23 which functions as a receptor for precursor proteins as they emerge from the TOM complex (Translocase of the Outer Membrane). Recently it was shown that Human Tim50, in addition to being component of the translocation machinery, exhibits phosphatase activity as a moonlighting function. However, it is not clear if this function influences processes within the cells. In another recent study, it was found that some mutations in the Human Tim50 gene are the cause of a mitochondrial disease, exhibiting epileptic encephalopathy. Initial investigation, in yeast, showed that these mutations do not affect protein import into mitochondria. We hypothesize, therefore, that these disease- causing mutations may affect the phosphatase activity of Human Tim50 or its interaction with other components in TIM23 complex. The objectives of the proposed research are to examine the phosphatase activity of Human Tim50 and the intercation with human Tim23, another component of the TIM23 complex.