Quantification of protein copy number in yeast: The NAD+metabolome

Abstract

Saccharomyces cerevisiae is calorie-Restricted by lowering glucose from 2% to 0.5%. Under low glucose conditions, replicative lifespan is extended in a manner that depends on the NAD+-Dependent protein lysine deacetylase Sir2 and NAD+salvage enzymes. Because NAD+is required for glucose utilization and Sir2 function, it was postulatedthat glucose levelsalter the levels of NAD+metabolites that tune Sir2 function. Though NAD+precursor vitamins, whichincrease the levels ofall NAD+metabolites, can extend yeast replicative lifespan, glucose restriction does not significantly change the levels orratios of intracellular NAD+metabolites. To test whether glucose restriction affects protein copynumbers, we developed a technology that combines the measurement of Urh1 specific activity and quantification of relative expression between Urh1 and any other protein. The technology was applied to obtain the protein copy numbers of enzymes involved in NAD+metabolism in rich and synthetic yeast media. Our data indicated that Sir2 and Pnc1, two enzymes that sequentially convert NAD+to nicotinamide and then to nicotinic acid, are up-Regulated by glucose restriction in rich media, and that Pnc1 alone is up-Regulated in synthetic media while levels of all other enzymes are unchanged. These data suggest that production or export of nicotinic acid might be a connection between NAD+and calorie restriction-Mediated lifespan extension in yeast.