Sphingolipids Control Lifespan Figure 1. The tetO7 promoter can be used to regulate CLS. (A) Model showing how sphingolipids are proposed to regulate lifespan. In this model LCBs and perhaps other sphingolipids activate the Pkh1 and Pkh2 protein kinases which phosphorylate residue T570 in the activation loop of the Sch9 protein kinase. Activation of Sch9 also requires phosphorylation of C-terminal residues by the TORC1 kinase. Ceramide-mediated activation of the Sit4 protein phosphatase may also play roles in regulating lifespan as outlined in the Discussion. (B) CLS of tetO7-SCH9 (RCD952) and WT (R1158) cells +/2 Dox treatment. Data show the mean 6 SEM of surviving cells (** p,0.01, No Dox vs Dox treatment). Triplicate cultures were used in these and all other CLS experiments. (C) CLS of tetO7-RIM15 (RCD994) and WT (R1158) cells +/2 Dox treatment. Data show the mean 6 SEM of surviving cells (* p,0.05, ** p,0.01, No Dox vs Dox treatment). (D) Oxidative stress (H2O2) resistance of cells from CLS day 1. Strains are: WT, R1158; tetO7-SCH9, RCD952; tetO7-RIM15, RCD994. doi:10.1371/journal.pgen.1002493.g001 (compared to the 72 hr time point for the other cultures). This corrected survival curve still shows an increase in lifespan (Figure 3B, dashed red line), but not as large an increase as the uncorrected curve (Figure 3B, solid red line). Myriocin treatment, similar to down-regulation of LCB1 or LCB2 expression, increases resistance of DBY746 cells to both heat and hydrogen peroxide stress (Figure 3G). In summary, the data presented in Figure 3 provide additional and independent support for the hypothesis that sphingolipids regulate stress resistance and CLS. Wild-type yeast cells normally grow to a specific size, but mutation of SCH9 has the unusual effect of reducing cell size [29]. If a reduction in sphingolipid synthesis increases CLS by reducing Sch9 activity as we predict (Figure 1A), then cell size ought to be reduced. We find that the volume of myriocin-treated DBY746 cells on CLS day 1 is 28% lower than that of untreated cells 3 (Figure S2 in Text S1, 59 verses 82 mm ). This reduction is less than the 40% reduction reported for sch9D cells compared to wild- type DBY746 cells [30], but probably reflects just a partial reduction of Sch9 activity in myriocin-treated cells (see below) compared to no activity in sch9D cells. Acetic acid accumulation in the diauxic shift has been proposed as a major cause of cell death, but it can be alleviated by buffering the medium [31]. To eliminate the possibility that myriocin treatment extends CLS by preventing acetic acid killing, we compared the effect of myriocin treatment in buffered and unbuffered medium. Medium was buffered with succinate (200 mM), pH 4.5, because this compound is not metabolized by yeast cells. Myriocin treatment extended CLS in either buffered or unbuffered media (Figure 3H). Thus, myriocin treatment does not extend lifespan simply by increasing resistance to acetic acid and additional support for this conclusion is provided below. Also, we found that the rate of cell death increased because the concentration of iron (1.23 mM) in SDC medium was limiting and we added 3-fold more iron to the medium (indicated in figure legends) to eliminate this variable. The succinate-buffered medium PLoS Genetics | www.plosgenetics.org 3 February 2012 | Volume 8 | Issue 2 | e1002493