P. G. Morgan, MD & M.M. Sedensky
The long-term goal of this work is to understand at the molecular level how mitochondrial function affects the aging process. We are studying two strains of C. elegans with mutations affecting mitochondrial function and which have altered lifespans. These mutant strains, clk-1 and gas-1, decrease the function of complex I of the electron transport chain. However, they differ in lifespan, sensitivity to oxygen, and accumulation of oxidative damage to mitochondrial proteins. The difference in free radical damage between the two strains is curious, since the mutations both affect the interaction of ubiquinone with complex I. The role of ubiquinone interaction with complex n has already been shown to play a role hi free radical formation with resulting effects on lifespan. The mutant clk-1 is deficient in the synthesis of ubiquinone, the electron acceptor for complexes I and ll. To survive, this mutant strain must obtain its ubiquinone from the bacteria on which it feeds. However, the ubiquinone which clk-1 obtains from the common form of E. coli used as food is different than the normal nematode ubiquinone (i.e. Q8 instead of Q9) and conveys a prolonged lifespan to the strain. We found that complex I and n differ in their ability to use the new ubiquinone; complex n functions normally while complex I is severely inhibited. The second mutation, gas-1, affects the 49 kDa subunit of complex I and forms part of the ubiquinone binding site of complex I. When we constructed the double mutant clk-1/gas-1 the resulting animals are sterile but live over twice as long as normal (34 days vs. 15 days). Thus, when gas-1 is exposed to Q8, it develops a prolonged lifespan. We hypothesize that the ubiquinone interaction with complex I directly affects respiration with secondary effects on ROS production. The specific aims of this application are to: 1. Measure the effects on respiration of substituting exogenous ubiquinones into mitochondria of N2, clk-1 and gas-1; 2. Determine the sites and rates of ROS production by mitochondria from N2, gas-1, and clk-1; 3. Demonstrate the resulting effects of the ubiquinones on oxidation of mitochondrial proteins; 4. Characterize two mutants, seg-1 and seg-2, that suppress the shortened lifespan of gas-1. These results will, for the first time, allow a correlation of the effects of ubiquinone on respiration with those on lifespan.