Sphingolipids Control Lifespan Dox vs pLCB1, 100 ng/ml Dox). (F) Control experiment showing that Dox-treatment extends CLS by reducing expression of the tetO7-LCB2 gene. CLS of tetO7-LCB2 cells transformed with a vector carrying LCB2 (pLCB2 = pRS315-LCB2-B7) or with an empty vector (Vec2 = pRS315) is shown. Data represent the mean 6 SEM of surviving cells (* p,0.05, ** p,0.01, for Vec2, 100 ng/ml Dox vs pLCB2, 100 ng/ml Dox). (G) Resistance of Dox-treated WT (R1158), tetO7-LCB1 (RCD956) and tetO7-LCB2 (RCD957) cells to heat (55uC) or hydrogen peroxide (H2O2) stress. Photographs show a ten-fold dilution series of cells (from left to right). doi:10.1371/journal.pgen.1002493.g002 may have a small affect on lifespan due to higher osmolarity as noted before [32], since we found that reducing the concentration from 200 to 50 mM caused a slight reduction in CLS, but no change in pH during the assay. The low pH of this medium is advantageous because the solubility of iron increases greatly at lower pHs [33]. Down-regulating LCB1 or SPT lowers sphingolipid levels If sphingolipids mediate increased CLS, then we predict that down-regulating tetO7-LCB1 expression by Dox treatment or reducing SPT activity by myriocin treatment should lower sphingolipid levels. We measured LCBs, made primarily as intermediates in the de novo sphingolipid synthesis pathway, as well as the terminal complex sphingolipids found primarily in the plasma membrane. We also measured LCBPs made by phosphorylation of LCBs and thought to primarily represent intermediates in the degradation of LCBs (Figure S1 in Text S1). We find that Dox treatment of tetO7-LCB1 cells, grown the same way as in a CLS assay, reduces total LCBs (C16-, C18- and C20- dihydrosphingosine and C18- and C20-phytosphingosine) by 84% with individual species of LCBs responding uniquely to drug treatment (Figure 4A). The much less abundant LCBPs are also reduced in toto by about 70% (Figure 4A). Dox treatment of wild- type R1158 cells does not produce a statistically significant change in the level of LCBs and LCBPs (data not shown). Similarly, myriocin treatment of DBY746 cells, grown in the same way as in a CLS assay, reduces total LCBs by 64% and total LCBPs 62% (Figure 4B). Next, we examined the effect of down-regulating sphingolipid synthesis on complex sphingolipids including inositol-phosphocer- amide (IPC), mannose-inositol-phosphoceramide (MIPC) and mannose-(inositol-P)2-ceramide (M(IP)2C) (Figure S1 in Text S1). There is a statistically significant reduction in IPCs in both Dox treated tetO7-LCB1 cells and myriocin-treated DBY746 cells (Figure 4C, 4D and 4E). Because MIPCs are not well separated from IPCs (Figure 4C), both types of sphingolipids, not just IPCs, may be reduced in drug-treated cells. There is a tendency for M(IP)2Cs to decrease in Dox-treated tetO7-LCB1 cells but to increase in myriocin-treated DBY746 cells. However, neither change is statistically significant, suggesting that M(IP)2Cs are not playing roles in CLS. Our data show that down-regulating the rate of sphingolipid synthesis by impairing activity of the first enzyme in the biosynthesis pathway lowers the concentration of LCBs, LCBPs, IPCs and perhaps MIPCs and some or all of these changes could influence CLS. Pkh2 regulates CLS As further support for our hypothesis that sphingolipids regulate CLS by controlling the Pkh1/2-Sch9 signaling pathway, we sought to show that PKH1 or PKH2 or both genes regulate CLS. Recently, deletion of PKH2 in the diploid BY4743 strain background was shown to produce a small but significant increase in CLS [34]. To verify these data and extend them to a haploid strain background and culture conditions with a different pH and buffering ions, we measured the CLS of a pkh2D mutant made in the R1158 strain background. We find a small but statistically significant increase in the CLS of pkh2D cells (Figure 5A). Our data are very similar to results obtained with BY4743 cells where the mean survival interval (area under a survival curve) was 3.10 for wild-type cells compared to 4.05 (34% increase) for pkh2D cells whereas our values are 4.14 verses 5.23 (29% increase). Together, the published data and our data establish a role for Pkh2 in regulating CLS. Phosphorylation of residue T570 in Sch9 is critical for protein kinase activity and is mediated in vivo and in vitro by Pkh1/2 [20,25] and is stimulated by LCBs [21]. If Pkh1/2 regulate Sch9 activity by phosphorylating residue T570, we anticipate less phosphorylation in pkh2D cells, since this would reflect reduced Sch9 activity and would explain the increase in CLS of pkh2D cells. We find a statistically significant 20% reduction of T570 phosphorylation in pkh2D cells compared to wild-type PKH2 cells which supports the hypothesis that Pkh2 phosphorylates this key Sch9 residue (Figure 5B and 5C). Another prediction of the proposed signaling pathway going from sphingolipids to Pkh1/2 to Sch9 and other downstream kinases (Figure 1A) is that growth of cells with a low level of Pkh kinase activity ought to be more sensitive to myriocin (reduced SPT activity) compared to cells with a higher level of kinase activity if sphingolipids regulate Pkh activity. Comparison of the myriocin sensitivity of cells with varying combinations of mutant and wild-type PKH1 and PKH2 alleles shows that cells with a temperature-sensitive PKH1 allele or a deletion allele are very sensitive to myriocin at a permissive temperature compared to cells with PKH1 or PKH2 or both wild-type genes (Figure 5D). Likewise, deletion of either gene in a tetO7-LCB1 strain reduces Pkh activity and increases sensitivity to myriocin (Figure S4A in Text S1), moreover, Dox treatment further reduces Pkh activity by lowering sphingolipid levels and impairs growth compared to the parent tetO7-LCB1 (PKH1 PKH2) strain (Figure S4B in Text S1). These data all support the proposed role of sphingolipids as upstream activators of the Pkh1/2 kinases. Sphingolipids control phosphorylation of Sch9-T570 and Sch9 substrates To explore further the hypothesized role of sphingolipids in regulating CLS via the Pkh1/2-Sch9 signaling pathway, we examined phosphorylation of Sch9 residue T570. If sphingolipids control T570 phosphorylation, then reducing SPT activity by myriocin treatment or by Dox treatment of tetO7-LCB1 cells should decrease phosphorylation. Cells were grown as in a CLS assay, but because drug-treated cells grow slower than untreated cells, comparisons were made with cells grown to similar A600 nm values (,1) rather than for similar times. We find a statistically significant 35% reduction in T570 phosphorylation in log phase DBY746 cells treated for 7–8 cell doublings with myriocin compared to untreated cells (Figure 5E and 5F, n = 4 cultures). Also, in parental R1158 (LCB1) and tetO7- LCB1 cells myriocin treatment reduces T570 phosphorylation by 40% (Figure 5G, compare lane 1 with 2 and lane 3 with 4). Lastly, there is a 40% reduction in T570 phosphorylation in Dox-treated tetO7-LCB1 cells (Figure 5H, compare lanes 3 and 4) but no effect in parental R1158 cells, as expected since LCB1 is not regulated by the tetO7 promoter (Figure 5H, compare lanes 1 and 2). These data PLoS Genetics | www.plosgenetics.org 5 February 2012 | Volume 8 | Issue 2 | e1002493