Using Saccharomyces cerevisiae as a Model to Investigate the Role of Human NAD Kinase

Heidi Hullinger.
Duke University

NAD kinase, the enzyme responsible for phosphorylating the cofactor NAD, is a component of multiple cellular processes, particularly fatty acid synthesis, the pentose phosphate pathway, and the reduction of the antioxidant glutathione. Since Saccharomyces cerevisiae has three genes encoding for NAD kinase, while humans only have one, each can be individually knocked out without loss of viability, making it an excellent model. One of the knockouts, pos5, has already been shown to increase the mitochondrial mutation rate in the cell. An increase in mitochondrial mutation rate has been linked to many neurodegenerative diseases and the aging process in human cells. I have thus been investigating whether human variants of the gene can complement the pos5 phenotype, which can be visually identified by a high frequency of petite colonies which have lost the ability to respire. While a vector containing the pos5 gene can rescue the knockout phenotype, cells containing a vector with the human version show petite frequencies similar to that of the knockout. Although I have identified a common polymorphism in the human gene, the variation has no effect on the observed petite frequency. The other two genes, UTR1 and YEL041w, do not seem to affect mitochondrial mutation rate, and I have not observed a phenotype for the double knockout. I am thus currently investigating the nuclear mutation rate by assaying the frequency of reversion in the knockouts. Not only does investigation of NAD kinase further our understanding of the metabolic processes it is involved in, but also may provide insight as to the effects of oxidative stress on the cell and its possible link to neurodegenerative disease and aging.