Sphingolipids Control Lifespan Figure 6. Down-regulating sphingolipid synthesis enhances CLS by Sch9-dependent mechanisms. (A) Doxycycline treatment enhances resistance of tetO7-LCB1 (RCD956) and tetO7-SCH9 (RCD952) cells to acetic acid. Cells from CLS day 1, grown in the presence or absence of Dox (300 ng/ml, were treated with acetic acid. (B) Myriocin treatment enhances resistance of DBY746 (WT) cells to acetic acid. Cells were grown and treated with myriocin (Myr) as described in (A). The sch9D strain was PF102. (C) The rate of oxygen consumption was measured in WT (DBY746) or sch9D (PF102) cells treated on not treated with myriocin (400 ng/ml). Cells were grown as described in panel B of Figure 5. Because cell volume is affected by myriocin treatment and deletion of SCH9, we measured cell volumes and normalized the values to the volume of 106 WT cells grown without myriocin treatment. Average values for four cultures and statistical significance are indicated. (D) Myriocin treatment (300 ng/ml) reduces the frequency of Canr mutations in sgs1D cells. Cells were grown in SDC medium (pH 4.5, 36 iron). Values represent the average for triplicate cultures started at an A600 nm of 0.05. Strains are: WT, RCD1009; sgs1D, RCD1010; sgs1D sch9D, RCD1011. (E) Myriocin treatment reduces the frequency of gross chromosomal rearrangements in sgs1D cells. Strains, culture conditions are the same as described in (D). doi:10.1371/journal.pgen.1002493.g006 Another group has also questioned the role of LCBs in activating Pkh1/2 kinase activity [27]. These authors found that short-term, acute dose myriocin treatment of cells failed to reduce Pkh1/2-mediated phosphorylation of residue T504 in the Ypk1 kinase. And recently, short-term, acute dose treatment of cells with myriocin did not decrease Pkh1/2-mediated phosphorylation of T570 in Sch9 [25]. In contrast to these results, we show that lowering the rate of sphingolipid synthesis by long-term, low dose myriocin treatment or down-regulation of LCB1 expression reduces Pkh1/2-mediated phosphorylation of T570 (Figure 5) and, in addition, myriocin treatment reduces phosphorylation of the Sch9 substrate Dot6 (Figure 5I). These results clearly support the idea that sphingolipids regulate lifespan by reducing Pkh1/2- mediated phosphorylation of T570 thereby reducing Sch9 activity. Further work is required to determine which sphingolipids control Sch9 activity and CLS (Figure 1A). Studies of the ISC1 gene also implicate sphingolipids in controlling lifespan. Isc1 hydrolyzes the polar head group from complex sphingolipids to yield ceramides. The CLS of isc1D cells is reduced partly because of mitochondrial dysfunctions [48], but CLS can be extended in these cells even beyond the wild-type value by deleting the SIT4 gene. Sit4 is the catalytic subunit of a PP2A-type protein phosphatase whose activity is down-regulated by TORC1 but up-regulated by ceramides (Figure 1A). In our experiments, enhancement of CLS by down-regulating sphingo- lipid synthesis in both wild-type and sch9D cells may involve Sit4, because reducing the abundance of IPCs, and perhaps MIPCs (Figure 4), may lower the amount of ceramide generated by Isc1. PLoS Genetics | www.plosgenetics.org 11 February 2012 | Volume 8 | Issue 2 | e1002493