Molecular mechanism of yeast chronological aging

Acetic acid is sufficient to cause chronological aging. (A) 2-day old BY4743 cultures were transferred to water adjusted to pH 2.8 with HCl and maintained at a concentration of 10 mM acetic acid. Acetic acid concentration was monitored every 1-2 hrs for the first 36 hours and additional acetic acid was provided as needed to maintain a concentration of 10 mM. The total molar amount of acetic acid added was 108.6 mM. (B) 2-day old BY4743 cultures grown in SC 0.05% were first lowered to pH 2.8 with HCl and then supplemented with 10 mM acetic acid. Acid was monitored similarly as (A) and added as needed over the first 36 hours. Mortality curve for SC 0.05% pH 2.8 + acetic acid was normalized for culture growth observed during the course of adding the acid by dividing the viability by the ratio of culture ODs (pH 2.8 + acetic acid/pH 2.8 alone). Error bars indicate the standard deviation of 3 biological replicates for untreated and pH 2.8 cultures and 6 biological replicates of pH 2.8 cultures maintained at 10 mM acetic acid.

Typically the chronological life span of yeast is measured by growing cells to saturation, plating a subset of the culture onto nutrient-rich YEPD plates, and counting the resulting colony forming units (CFUs) over time. We have developed a novel high-throughput method for measuring the chronological life span of the yeast Saccharomyces cerevisia that calculates viability of aged cells by measuring the shift in growth curves over a set time course. This method retains the same accuracy in measuring viability as the CFU method does while greatly increasing the potential data output. You can read more about our method here. In addition, we are in the process of creating a website open for anyone to use that greatly simplifies data analysis when using this method.

Using this method, we recently found that growth in standard synthetic defined medium results in an acidification of the culture supernatant which correlates with a reduction in the chronological life span of the budding yeast Saccharomyces cerevisia. Dietary restriction, growth in a non-fermentable carbon source such as glycerol, or transferring the cells to water results in lower levels of acidic byproducts in the yeast cultures. Further tests identified acetic acid as a cell-extrinsic mediator of cell death during the life span (see figure, taken from Burtner et al., Cell Cycle, 2009). In addition, several long-lived mutant s including sch9Δ and ras2Δ cells showed an increased resistance to acetic acid. These results indicate the extracellular environment plays a significant role in determining the chronological life span of yeast.

To follow up on this idea, we have performed a screen of the ORF deletion collection to identify single gene deletion mutants that do not acidify their supernatant to the same degree as wild-type cells. Several mutants have been found that have a higher culture pH as well as being significantly longer lived than wild-type cells and are being investigated further.

People: Chris Murakami