Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast

Abstract

Our understanding of sphingolipid metabolism and functions in the baker's yeast Saccharomyces cerevisiae has progressed substantially in the past 2 years. Yeast sphingolipids contain a C26-acyl moiety, all of the genes necessary to make these long-chain fatty acids have been identified, and a mechanism for how chain length is determined has been proposed. Advances in understanding how the de novo synthesis of ceramide and complex sphingolipids is regulated have been made, and they demonstrate that the Target Of Rapamycin Complex 2 (TORC2) controls ceramide synthase activity. Other work shows that TORC2 regulates the level of complex sphingolipids in a pathway using the Slm1 and Slm2 proteins to control the protein phosphatase calcineurin, which regulates the breakdown of complex sphingolipids. The activity of Slm1 and Slm2 has also been shown to be regulated during heat stress by phosphoinositides and TORC2, along with sphingoid long-chain bases and the Pkh1 and Pkh2 protein kinases, to control the actin cytoskeleton, the trafficking of nutrient transporters, and cell viability. Together, these results provide the first molecular insights into understanding previous genetic interaction data that indicated a connection between sphingolipids and the TORC2 and phosphoinositide signaling networks. This new knowledge provides a foundation for greatly advancing our understanding of sphingolipid biology in yeast.

Fig. 1.

Outline of sphingolipid metabolism in Saccharomyces cerevisiae. Metabolic intermediates and complex sphingolipids are shown in boldface, genes are indicated by italics, and enzyme names are in regular lettering. Structures of the indicated compounds are presented in previous publications (9, 14). The cellular locations of these reactions are discussed in the text and in a recent discussion of intracellular sphingolipid trafficking (106). When grown aerobically, the fatty acid in complex sphingolipids is often hydroxylated at C2 and sometimes at C3, a reaction that requires Scs7 (not shown). Ceramides can be hydrolyzed by two ceramidases, Ydc1 and Ypc1, to yield a fatty acid and a long-chain base (LCB) (107, 108).

Fig. 2.

Target Of Rapamycin Complex 2 (TORC2) and calcineurin regulate de novo ceramide and sphingolipid synthesis in yeast. TORC2 is proposed to sense growth signals and phosphorylate the protein kinase Ypk2 (61), which, after being phosphorylated in its activation loop by Pkh2 (63) (and perhaps Pkh1), activates ceramide synthase. The Ca²⁺/calmodulin-regulated protein phosphatase calcineurin opposes the TORC2-Pkh1/2-Ypk2 pathway to downregulate ceramide synthase activity in response to heat and other stresses. Environmental factors such as heat stress that increase LCBs are proposed to act in a feed-forward manner to activate Pkh1/2 and possibly increase the rate of ceramide synthesis (not shown) (61).

Fig. 3.

Summary of the signaling pathways regulated by LCBs in S. cerevisiae. Heat stress is known to produce a transient increase in LCBs, which then activate Pkh1 and Pkh2. In vitro, Pkh2 is more strongly stimulated by LCBs than is Pkh1 (66), but the situation in vivo has not been analyzed directly by enzyme activity measurements. Studies in vitro suggest that LCBs can directly produce small increases in the activity of Ypk1, Ypk2, and Sch9 (indicated by dotted lines), whereas the stimulation of Pkc1 has not been examined (66). Pkh1/2 phosphorylate Ypk1, Ypk2, Sch9, and Pkc1 in their activation loop (PDK1 site), but the proteins are not enzymatically active. To become active, they also need to be phosphorylated in a hydrophobic region (PDK2 site) and in a turn motif in their C termini. Phosphorylation of these sites in Ypk2 is mediated by TORC2 (63), and for Sch9 phosphorylation is mediated by TORC1 (109). Ypk1/2 and Pkc1 are shown working in parallel pathways to control cell wall integrity, but data also support an alternative pathway in which Ypk1/2 work upstream of Pkc1 (75, 76).

Fig. 4.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P₂) and the Slm1 and Slm2 proteins regulate the synthesis and turnover of complex sphingolipids and the dynamics of the actin cytoskeleton. The enzymes Stt4 and Mss4, which synthesize PI4,5P₂ along with the TORC2 protein kinase, are proposed to activate Slm1 and Slm2, which then downregulate the turnover of complex sphingolipids, particularly inositol phosphoceramide (IPC), by the Isc1 enzyme. The Slm proteins also regulate the calcium/calmodulin-regulated protein phosphatase calcineurin, which dephosphorylates and inactivates the Slm proteins and also interacts with Csg1/2 in an unknown manner to regulate the conversion of IPC to mannose inositol phosphoceramide by the Csg1/2 enzymes. Slm regulation of the Rho1/Pkc1 pathway is independent from the regulation of sphingolipid metabolism. Genetic interactions suggest that IPC plays a role in actin organization, but the mechanism is unknown. PI, phosphatidylinositol; PI4P, phosphatidylinositol 4-phosphate.