LETTERS Plasma membrane stress induces relocalization of Slm proteins and activation of TORC2 to promote sphingolipid synthesis Doris Berchtold 1,7 , Manuele Piccolis 2,7 , Nicolas Chiaruttini 3 , Isabelle Riezman 3 , Howard Riezman 3,4 , Aurélien Roux 3,4 , Tobias C. Walther 1,5,8,9 and Robbie Loewith 2,4,6,8,9 The plasma membrane delimits the cell, and its integrity is essential for cell survival. Lipids and proteins form domains of distinct composition within the plasma membrane. How changes in plasma membrane composition are perceived, and how the abundance of lipids in the plasma membrane is regulated to balance changing needs remains largely unknown. Here, we show that the Slm1/2 paralogues and the target of rapamycin kinase complex 2 (TORC2) play a central role in this regulation. Membrane stress, induced by either inhibition of sphingolipid metabolism or by mechanically stretching the plasma membrane, redistributes Slm proteins between distinct plasma membrane domains. This increases Slm protein association with and activation of TORC2, which is restricted to the domain known as the membrane compartment containing TORC2 (MCT; ref. 1). As TORC2 regulates sphingolipid metabolism 2 , our discoveries reveal a homeostasis mechanism in which TORC2 responds to plasma membrane stress to mediate compensatory changes in cellular lipid synthesis and hence modulates the composition of the plasma membrane. The components of this pathway and their involvement in signalling after membrane stretch are evolutionarily conserved. Sphingolipids localize mostly in the outer leaflet of the plasma membrane and play a role in maintaining its barrier function 3,4 . Sphingolipid abundance is controlled by feedback mechanisms that regulate the activity of sphingolipid biosynthetic enzymes to achieve homeostasis of lipid composition in the membrane 5 . The sensing mechanism detecting changes of sphingolipids in the plasma membrane is yet unknown. The evolutionarily conserved target of rapamycin (TOR) kinase stimulates cell mass accumulation as part of two multi-protein 1 Max Planck Institute of Biochemistry, Organelle Architecture and Dynamics, 82152 Martinsried, Germany. 2 Department of Molecular Biology, University of Geneva, Geneva 1211, Switzerland. 3 Department of Biochemistry, University of Geneva, Geneva 1211, Switzerland. 4 Swiss National Centre for Competence in Research Programme Chemical Biology, Geneva 1211, Switzerland. 5 Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. 6 Swiss National Centre for Competence in Research Programme Frontiers in Genetics, Geneva 1211, Switzerland. 7 These authors contributed equally to this work. 8 These authors contributed equally to this work. 9 Correspondence should be addressed to T.C.W. or R.L. (e-mail: tobias.walther@yale.edu or robbie.loewith@unige.ch) Received 30 January 2011; accepted 12 March 2012; published online 15 April 2012; DOI: 10.1038/ncb2480 complexes named TOR complex 1 (TORC1) and TORC2 (ref. 6). The rapamycin-sensitive TORC1 regulates and is regulated by ribosome biogenesis in a product inhibition feedback loop 7 . The rapamycin- insensitive TORC2 supports growth by enhancing sphingolipid synthesis 2 , but the regulation of this kinase complex, through feedback loops or otherwise, is unknown, although a combination of sterol and sphingolipid defects has been shown to reduce TORC2 activity 8 , implicating a control through lipid homeostasis. To assess TORC2 activity, and thus begin to determine whether it is regulated by sphingolipid levels, we measured the signal intensity on western blots probed with an antiserum generated to detect phosphorylation of the TORC2 target site T662 of the kinase Ypk1 (Fig. 1a and Supplementary Fig. S1a–c). Ypk1 is one of two paralogues crucial for transduction of TORC2 signals to regulate sphingolipid synthesis 2 . The sphingolipid synthesis pathway of yeast 9 is amenable to genetic and pharmaceutical inhibition (Fig. 1b). Myriocin (Myr) inhibits serine palmitoyltransferase (SPT) catalysing the first and committing step of sphingolipid synthesis and treatment of cells strongly reduced phytoceramide (PHC) levels, as well as inositolphosphoceramide (IPC) and mannosyl-inositolphosphoceramide (MIPC) levels within minutes. In contrast, aureobasidin A (AbA), which inhibits synthesis of complex sphingolipids, reduced only IPC and MIPC levels within this time frame. Neither drug strongly affected the levels of mannosyl- di-inositolphosphoceramide (M(IP) 2 C) (Fig. 1c). Both Myr and AbA stimulated TORC2 activity, evident by increased levels of Ypk1 T662 phosphorylation (Fig. 1d), whereas TORC1 activity, as assessed by monitoring the TORC1-specific hydrophobic motif phosphorylation of Sch9 (ref. 10,11), was unaltered by these treatments (Fig. 1d and Supplementary Fig. S1c,d). The Myr- or AbA-induced increase of Ypk1 T662 phosphorylation was specifically mediated by TORC2 (Fig. 1e) and not by inhibition of a phosphatase (Supplementary NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1