|Budget Amount *help
¥7,500,000 (Direct Cost : ¥7,500,000)
Fiscal Year 1997 : ¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1996 : ¥4,800,000 (Direct Cost : ¥4,800,000)
Most of mitochondrial proteins are synthesized as precursor proteins in the cytosol and imported into mitochondria with the aid of protein translocation machineries in the outer and the inner membranes called the TOM complex and the TIM complex, respectively, In the present study, we analyzed subunit arrangements, structures, and precursor interactions of the mitochondrial translocation machineries.
The TOM complex consists of receptor proteins, Tom7O, Tom37, Tom2O and Tom22, a channel protein, Tom4O, and small Tom proteins, Tom5, Tom6 and Tom7, By using differential solubilization methods, we found that Tom7OiTom37 and Tom2O/Tom22/Tom4O form distinct subcomplex structures in the TOM complex. We prepared cytosolic domains of Tom7O and Tom22 in large amounts and analyzed Tom7O-Tom2O interactions by glycerol gradient centrifugation. The results showed that a positively charged segment just downstream of the N-terminal transmembrane segment of Tom22 is important for interactions of Tom2O with possibly a negatively charged domain of Tom7O.Next, a tertiary structure of the cytosolic domain of Tom2O was determined by NMR spectroscopy. A binding site for presequences is also mapped on the NMR structure. In the last, we introduced a photoreactive unnatural amino acids into various positions along mitochondrial precursor protein by suppressor *RNA method, By using these labeled precursor proteins, interactions of the translocating polypeptide chains with components of the mitochondrial translocation machineries were analyzed by photocrosslinking at a high resolution that had not been obtained so far.