The protein kinase Sch 9 is a key regulator

The Saccharomyces cerevisiae protein kinase Sch9 has been demonstrated to be an in vitro and in vivo effector of sphingolipid signalling. In this study, the link between Sch9 and sphingolipid metabolism in S. cerevisiae was examined in vivo based on the observation that the sch9Δ mutant displays altered sensitivities to various inhibitors of sphingolipid metabolism, i.e. myriocin and aureobasidin A. Sphingolipid profiling indicated that sch9 cells have increased levels of long-chain bases and long-chain base-1 phosphates, decreased levels of several species of (phyto)ceramides, and altered ratios of complex sphingolipids. We show that the TORC1 – Sch9 signalling pathway functions to repress the expression of the ceramidase genes YDC1 and YPC1, thereby unveiling, for the first time in yeast, a nutrient-dependent transcriptional mechanism involved in the regulation of sphingolipid metabolism. Additionally, we established that Sch9 affects the activity of the inositol phosphosphingolipid phospholipase C, Isc1, which is required for ceramide production by hydrolysis of complex sphingolipids. As sphingolipid metabolites play a crucial role in the regulation of stress tolerance and longevity of yeast cells, our data provide a model in which Sch9 regulates the latter phenotypes by acting not only as an effector but also as a regulator of sphingolipid metabolism. http://www.molbiolcell.org/content/suppl/2013/11/04/mbc.E13-06-0340v1.DC1 Supplemental Material can be found at: 2 INTRODUCTION Sphingolipids, together with sterols and glycerophospholipids, are essential components of all eukaryotic membranes. Much of the progress in the understanding of sphingolipid metabolism is due to research performed in the budding yeast Saccharomyces cerevisiae, in which most, if not all, of the enzymes involved in sphingolipid metabolism have been identified (Figure 1) (Funato et al., 2002; Sims et al., 2004; Dickson et al., 2006). Apart from their structural function, sphingolipids also play important regulatory roles. More specifically, in yeast, sphingolipids were shown to be involved in the regulation of cellular processes as diverse as cell growth, endocytosis, actin cytoskeleton organization, protein trafficking, cell wall integrity, nutrient uptake and longevity (Dickson, 2010). In these processes, signalling functions are not only ascribed to the complex sphingolipid species, but also to the intermediate metabolites, i.e. the long-chain bases (LCBs), the long-chain base -1-phosphates (LCBPs) and ceramide species. Accumulating evidence in yeast and mammalian model systems strongly suggest that the dynamic balance between the different sphingolipid metabolites, often referred to as the LCB(P)/ceramide rheostat or sphingolipid rheostat, is an important factor determining their regulatory action (Kobayashi and Nagiec, 2003; Kihara et al., 2007; Dickson, 2008; Matmati and Hannun, 2008; Breslow and Weissman, 2010; Bikman and Summers, 2011; Van Brocklyn and Williams, 2012). As such, proper regulation of this balance is key to cellular survival, and recent reports have shed some light on the molecular mechanisms by which yeast controls this balance. For instance, the yeast Orm1 and Orm2 proteins were demonstrated as crucial regulators of sphingolipid homeostasis by controlling the activity of the Serine Palmitoyl Transferase (SPT) enzyme, which catalyzes the first and rate limiting step in the de novo synthesis of sphingolipids. Orm1/2 are members of the conserved ORMDL family of endoplasmic reticulum (ER) membrane proteins (ORMDL 1/2/3 genes in humans), and were shown to physically interact with SPT, thereby inhibiting the activity of the enzyme (Breslow et al., 2010; Han et al., 2010). This inhibitory action was shown to be abrogated through Orm1/2 phosphorylation by the protein kinase Ypk1 (Roelants et al., 2011). When sphingolipid levels drop, e.g. when cells were treated with the SPT inhibitor myriocin, the TORC2 kinase will activate Ypk1, thereby boosting de novo sphingolipid synthesis to counteract dropping sphingolipid levels. In addition, the transient increase in LCB accumulation upon heat shock was also shown to be dependent on Ypk1’s action, downstream of the Pkh1/2 kinases (Sun et al., 2012). Interestingly, as yeast cells approach stationary phase, they accumulate LCBs and LCBPs as well (Lester et al., 2013). This increase may also be mediated in part by an increase in SPT activity, though additional mechanisms, such as a decrease in ceramide synthase activity as cells enter stationary phase, were also demonstrated. Although the Orm proteins were shown to be phosphorylated by the rapamycin-sensitive, nutrient-sensitive TORC1 kinase, recent evidence revealed that these phosphorylation sites were distinct from those regulated by myriocin treatment, indicating that TORC1 and TORC2 have distinct role in the control of the Orm proteins (Liu et al., 2012;