Jcb_201606059 131..148

Several critical lipid intermediates, such as DAG and ceramide, can be toxic when they accumulate in cellular membranes. This is not only because they affect membrane structure and organization but also because they are potent signaling molecules. Numerous studies have linked elevated ceramides with cellular stress, cell cycle arrest, apoptosis, and insulin resistance; ceramide accumulation is also associated some cancers and neurodegenerative diseases (Xie et al., 1998; Holland et al., 2007; Pickersgill et al., 2007; Ledeen and Wu, 2008; Bikman and Summers, 2011; Mullen and Obeid, 2012). Ceramide toxicity has also been demonstrated in Saccharomyces cerevisiae and may cause an apoptosis-like cell death (Eisenberg and Büttner, 2014). How cells monitor ceramide levels and prevent the accumulation of excess ceramide is only partially understood. Ceramides are synthesized de novo in ER membranes by the N-acylation of sphingoid long-chain bases (LCBs) with fatty acids (Fig. 1 A; Merrill, 2002). They are also generated by degradation of complex sphingolipids. Ceramide levels are modulated by mechanisms in addition to regulating synthesis. Most cells possess several ceramidases, which hydrolyze ceramides into LCBs and fatty acids (Ito et al., 2014). There is also growing evidence that cells convert excess ceramides into other lipids to prevent the toxic accumulation of ceramides. For example, a sphingomyelin synthase-related protein in the ER generates a sphingomyelin analogue from ceramide (Ternes et al., 2009; Vacaru et al., 2009), and it has recently been found that this enzyme is necessary to prevent the toxic accumulation of ceramides and apoptosis (Tafesse et al., 2014). Another method for removing excess ceramide has been identified in yeast. It has recently been found that ceramides can be converted into acylceramides, which are probably not toxic (Voynova et al., 2012). How ceramides traffic in cells is only incompletely understood. After ceramides are synthesized in the ER, they are transferred to the Golgi complex, which is the location of the enzymes that generate complex sphinogolipids from ceramide. Ceramide transport from the ER to the Golgi complex occurs by both vesicular and nonvesicular mechanisms. Ceramide transport protein (CERT) facilitates nonvesicular ceramide transport in mammalian cells; cells that lack this protein have a significantly reduced rate of sphingomyelin formation (Hanada et al., 2003). In S. cerevisiae, ceramides are also transported from the ER to the Golgi complex by both vesicular and nonvesicular pathways (Funato and Riezman, 2001). The nonvesicular pathway seems to be relatively minor in this yeast because mutations that block vesicular trafficking reduce ceramide transport by ∼80% (Funato and Riezman, 2001). S. cerevisiae lacks a CERT homologue, and it is not known how nonvesicular transport is facilitated. Once ceramides reach the medial-Golgi in Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess cera‐ mide is toxic, causing growth arrest and apoptosis. In this study, we identify a novel mechanism by which cells prevent the toxic accumulation of ceramides; they facilitate nonvesicular ceramide transfer from the endoplasmic reticulum (ER) to the Golgi complex, where ceramides are converted to complex sphingolipids. We find that the yeast protein Nvj2p promotes the nonvesicular transfer of ceramides from the ER to the Golgi complex. The protein is a tether that generates close contacts between these compartments and may directly transport ceramide. Nvj2p normally resides at contacts between the ER and other organelles, but during ER stress, it relocalizes to and increases ER–Golgi contacts. ER–Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER–Golgi con‐ tacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the buildup of toxic amounts of ceramides. An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity