Pathway analysis of ethanol and acetic acid metabolism in Drosophila. K.L. Montooth , A.G. Clark. Department of Biology, Pennsylvania State University, University Park, PA.

   Ethanol and acetic acid are important environmental stresses for species of Drosophila that consume fermenting fruit. Survival tolerances for both compounds are highly positively correlated across populations of D. melanogaster and species of Drosophila (Chakir et al. 1993), and short-term exposure to either results in a similar metabolic response (Clark 1997; Clark and Fucito 1998). The non-independence of traits associated with different steps in the metabolism of ethanol and acetic acid underscores the need for an analysis of the complete metabolic pathway from ethanol to acetyl CoA. A pathway analysis has been hindered by the fact that only alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) could be studied at the biochemical and molecular levels, leaving the third step in the pathway, catalyzed by acetyl-CoA synthetase (AcCoAS), uncharacterized. Identification of AcCoAS from the complete genome sequence of D. melanogaster and recently developed molecular probes for the AcCoAS reaction now allow for a complete pathway analysis. We hypothesize that selection for increased ethanol and acetic acid tolerance operates on properties of the entire metabolic pathway. We predict that ethanol and acetic acid tolerance will be better modeled using information from all three steps in the pathway rather than by looking for simple relationships between a single enzyme and tolerance. Here we report findings from a study quantifying variation and covariation among ADH, ALDH and AcCoAS enzyme kinetics, mRNA levels, and ethanol and acetic acid tolerance in isofemale lines of D. melanogaster from 3 populations and in D. mauritiana, D. sechellia, D. simulans, and D. yakuba. Properties of the entire pathway, including flux and inferred relative metabolite concentrations, were calculated from measures of enzyme kinetics at all three steps in the pathway. We then use these global properties of the pathway to predict ethanol and acetic acid survival tolerance.