Worm Breeder's Gazette 17(1): 68 (October 1, 2001)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Mitochondrial Metabolism in C. elegans

Marge Sedensky1, Phil Morgan2

1 University Hospitals and CWRU Cleveland Ohio
2 "University Hospitals and CWRU Cleveland Ohio

We have been studying a mutation called gas-1 that was isolated in a screen for hypersensitivity to the volatile anesthetic isoflurane. This animal is hypersensitive to all the volatile anesthetics in which it has been tested, has a shortened lifespan, produces only 50 or so eggs at room temperature, and is basically a temperature sensitive lethal. gas-1 encodes a subunit of the first protein complex of the electron transport chain. We>have done a great deal of metabolic measurements to characterize the defect in this animal. The ability of mitochondria from gas-1 to metabolize complex I specific substrates like malate or glutamate is about 1/4 that of N2 mitochondria. Their ability to use succinate, a substrate specific for complex II, is greater than N2. gas-1 animals possess the same ratio of ATP to ADP + AMP as N2 when in air, but this ratio drops when they are immobilized in a volatile anesthetic. We are now mapping two suppressors of the anesthetic sensitive phenotype. These two mutations are dominant, with no clear phenotype of their own for mapping. The suppressors do extend the lifespan of gas-1, although they do not restore it to normal. This is also true for the ability of their mitochondria to metabolize malate or glutamate.

We have now produced an antibody to GAS-1 that is designed to distinguish it from another protein that may be expressed from a virtually identical cDNA that is contained on the cosmid T26A5.3. Although we know this sequence is expressed, we have not seen any signal via a GFP promoter, whereas a gas-1 construct apppears widely expressed. T26A5.3 does not rescue gas-1 unless the sequence is placed under the gas-1 promoter. We are also trying to identify the different complexes of the electron transport chain via a native gel in order to see the stoichiometry of the subunits of complex I in a gas-1 animal. Although we can estimate weights of each complex based on the predictions of genomic sequences of each subunit of each complex, commercial antibodies so far have not helped us distinguish Complex I from the other four major bands on gels. Another major effort on this project is the identification of the kinds of damage generated by defective mitochondria in gas-1 animals. Westerns that are probed with an antibody to products of lipid peroxidation and also gels using a kit that measures oxidative damage to proteins show that gas-1 mitochondria undergo more oxidative damage than N2. We are planning to identify the major proteins damaged in gas-1 mitochondria. Since we are anesthesiologists, we have also been interested in patients with mitochondiral myopathies. There have long been anecdotal reports of children with mitochondrial defects that are hypersensitive to anesthetics. We have some observations on children that have come to our operating room for either diagnosis or treatment of a mitochondrial disease, that would suggest that different defects may lead to different sensitivities to a volatile anesthetic. Only those few children we observed with a complex I defect were hypersensitive (4 children out of 16 patients). Children with defects in Complex II were not hypersensitive. We know that mev-1, a defect in Complex II in C. elegans, shares many phenotypes with gas-1, but is not hypersensitve to volatile anesthetics. It cannot use succinate as a substrate for oxygen consumption. Isolation of mitochondria, measurements of ATP, extraction of mitochondrial proteins, and attempts to age-match very large numbers of mutant worms have also absolutely demonstrated to us the reasons biochemistry is not much fun in C. elegans. Once in a while we do a nice mating just to satisfy a longing for the good old days.