Mary B. Kroetz
Department of Biology
Marquette University

The F1F0-ATP synthase is the enzyme responsible for the aerobic synthesis of ATP in all living eukaryotes. Recent studies show that in the yeast Saccharomyces ceervisiae the F1F0-ATP synthase exists as a dimmer in the mitochondria and subunits e, g, and k associate with this dimeric complex. Characterization of these three subunits has shown that they are not essential for the function of the F1F0-ATP synthase. Deletion of the genes encoding subunit c and g individually in a haploid yeast strain however, affected the ability of the F1F0-ATP synthase to form dimmers. The enzyme was present in its monomeric form in these null mutant strains, delta su 3 and delta su g, respectively. Deletion of the gene for subunit k (delta su k) had no apparent effect on the activity or the dimerization of the ATP synthase at 30oC. Recent studies have shown however, that the delta su k null mutant displays a growth phenotype at the elevated temperature of 37oC on a nonfermentable carbon source. This growth phenotype indicates that oxidative phosphorylation is affected in the delta su k at elevated temperatures.

My research interest is two-fold. To date, yeast strains exist where the genes encoding subunits e, g, and k have been deleted or “knocked-out” individually. My first project was to create so-called “double-knock-outs”, where two of the genes encoding these subunits have been deleted in the same cell. A technique termed PCR-based gene disruption was used to achieve this goal. Once the double-knock-outs are obtained, the growth phenotype of these mutants on non-fermentable carbon sources can be characterized. Both enzyme activities and the protein levels of various subunits of the F1F0-ATP synthase, as well as the electron transport chain complexes will be determined. The second area of interest focuses on the temperature sensitive phenotype of the null mutant, delta su k. To determine whether the observed phenotype was directly due to the loss of subunit k, the mutant was transformed with a plasmid containing the gene for the subunit k in an attempt to rescue the phenotype. These projects will be continued in the fall to obtain further results.