The burgeoning field of bioremediation relies on the natural abilities of plants and fungi to accumulate certain toxic heavy metals. In heavy metal detoxification, plant and yeast vacuoles are responsible for the sequestration of toxins away from the cytoplasm. A yet-unpublished study done by the Rea group analyzed the protein profile of the vacuolar lumen in the budding yeast Saccharomyces cerevisiae. Several NAD+-dependent dehydrogenases were found within this compartment, a surprising finding in light of the yeast vacuole's predominantly lytic function. Five of these enzymes were found to increase in level in the vacuole during heavy metal stress. Moreover, when vacuolar lysates were assayed in vitro, they were found to contain dehydrogenase activity when exogenous NAD+ or NADH was provided. If these enzymes are also active in this compartment in vivo, the question is raised: from where do the cofactors required for the reactions that these enzymes catalyze come? If these enzymes had a vacuolar source of NAD+, they could also potentially be active in vivo. Thus, in the present project, we are seeking to determine the concentration of NAD+ inside the vacuole of S. cerevisiae. Toward this end, high-purity "proteomics-grade" intact vacuoles were isolated from S. cerevisiae by a combination of differential, density, and floatation centrifugation. NAD+ was then extracted by acid precipitation and solvent extraction to separate vacuolar proteins from cofactor. NAD+ was quantified using a two-step redox-coupled reaction system containing phenazine methosulfate (PMS) as mediator and thiazoyl blue tetrazolium bromide (MTT) as terminal electron acceptor. The spectrophotometric measurement of reduced MTT at 570 nm is an indirect measure of the initial NAD+ concentration. To provide a basis for comparison, the estimated NAD+ content of isolated vacuoles was compared to that of spheroplasts lysates extracted and assayed identically. The results indicate that the intravacuolar concentration of NAD+ is two orders of magnitude lower than that of the spheroplast (5.2 vs. 202 μM), which may have implications for this redox-active cofactor’s function in the vacuole. These findings may necessitate the reconsideration of the role played by vacuolar dehydrogenases in yeast cell metabolism (and possibly the metabolism of other vacuolate cells). Our findings suggest that the vacuolar pool of NAD+ may be sufficient for utilization in the vacuole.
"Searching for the Source: Determining NAD+ Concentrations in the Yeast Vacuole,"
Penn McNair Research Journal: Vol. 1
, Article 2.
Available at: http://repository.upenn.edu/mcnair_scholars/vol1/iss1/2