Download endoplasmic reticulum stress and lipid metabolism

ENDOPLASMIC RETICULUM STRESS AND LIPID METABOLISM MIKE F. RENNE1
ABSTRACT Upon endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) triggers cellular mechanisms to restore ER homeostasis. Aberrancies in lipid homeostasis can cause alterations in biochemical and biophysical properties of the ER membrane, which impair ER function and induce UPR signaling. The UPR is also involved in regulation of lipid metabolism and membrane biogenesis, indicating a link between ER-­‐stress and lipid homeostasis. In this review, we will discuss how alterations in lipid metabolism can activate the UPR, as well as how the UPR alters lipid metabolism to relieve stress from the ER. INTRODUCTION The endoplasmic reticulum is part of the endomembrane system, which further consists of the nuclear envelope, plasma membrane (PM), Golgi apparatus, endosomes, lysosomes, lipid droplets, peroxisomes and secretory vesicles. The ER is the entry point to the secretory pathway, and plays a central role in the synthesis of proteins but is also the site of phospholipid biosynthesis. It is responsible for all de novo synthesis of membranes within the cell and has a dynamic membrane structure that allows fast expansion to accommodate protein and membrane biosynthesis. The ER consists of the cisternal ER (historically referred to as “rough ER”) and tubular ER (historically referred to as “smooth ER”). Both compartments of the ER are 1
specialized for different functions and therefore differ in form. The cisternal ER is continuous with the nuclear envelope and is studded with ribosomes, thus the cisternal sheets are the location of protein synthesis and folding. The tubular ER is enriched in tissues specializing in the biosynthesis of lipids and steroids and the large amount of enzymes required for these processes. In addition to anterograde or retrograde vesicular transport, the ER can form membrane junctions, or contact sites, with other organelles to facilitate inter-­‐organelle trafficking of lipids, enzymes and other compounds. When something goes wrong in cellular homeostasis, such as the accumulation of unfolded or misfolded proteins due to inherited mutations, calcium-­‐ or oxidative flux, the cell can be under (ER) stress. In 1988, a stress response to unfolded proteins was reported, which we now know as the unfolded protein response (UPR) (Kozutsumi, Segal, Normington, Gething, & Sambrook, 1988). ER stress is synonymous with the activation of the UPR. The key signaling proteins for the UPR are ER-­‐localised transmembrane proteins that communicate perturbations within the lumen to initiate downstream cytoplasmic signaling cascades that function to resolve stress or, when in crisis, signal apoptosis. Chronic ER stress has therefore been implicated in the pathogenesis of many degenerative diseases associated with lipid metabolism, Membrane B iochemistry & Biophysics, Bijvoet Centre for Biomolecular Research and Institute of Biomembranes, Utrecht University, the Netherlands including metabolic diseases (e.g. diabetes, obesity) and inflammatory diseases (e.g. artherosclerosis). While cytosolic enzymes execute the synthesis of fatty acids, the elongation of fatty acids and the assembly of lipids occur predominantly in the ER. This review will discuss studies from both mammalian systems and the model eukaryote Saccharomyces cerevisiae (baker’s yeast or yeast in short). Yeast is a widely used model eukoryote in studies on ER-­‐related subjects, such as ER-­‐stress and lipid metabolism. Lipid metabolism and regulation are highly conserved between yeast and mammals (Nielsen, 2009). Furthermore, the yeast UPR is conserved in one of the branches of the mammalian UPR, as well as one of the mammalian UPR branch is highly homologues to the yeast UPR (Mori, 2009). There is accumulating evidence, from yeast and mammalian model systems, that loss of lipid homeostasis causes ER stress and that the UPR is an important sensor of changes in ER membrane homeostasis and is required for lipid biosynthesis. Therefore, this review will discuss the intersection between the UPR and lipid metabolism. LIPID METABOLISM AT A GLANCE Membrane lipids can be discriminated on the base of their chemical structure, dividing them into three main families: glycerophospholipids (phospholipids, PL), glycerosphingolipids (sphingolipids, SL) and sterols. Within these three families, there are various different classes in which thousands of different molecular species have been identified. The lipid composition of the membrane determines the biophysical properties essential for proper membrane (and membrane protein) function, such as surface charge, thickness and fluidity of the membrane (reviewed by de Kroon, Rijken, and De Smet (2013)). De novo synthesis of lipids starts with the formation of acetyl-­‐
coenzyme A (Acetyl-­‐CoA) in the cytosol. To synthesize of fatty acids, acetyl-­‐CoA is carboxylated to malonyl-­‐CoA. Fatty acid synthases synthesize (mainly) palmitoyl-­‐
CoA from malonyl-­‐CoA and acetyl-­‐CoA; palmitoyl-­‐CoA can be processed (elongation, desaturation) in the ER to provide different fatty acids bound to CoA (Acyl-­‐CoA) (Figure 1). In the following section we will briefly discuss the synthesis of lipids in mammals and yeast. For a more complete coverage of these topics, the reader is referred to recent reviews (Henry, Kohlwein, & Carman, 2012; Holthuis & Menon, 2014; Nohturfft & Zhang, 2009) and references therein. PHOSPHOLIPIDS The bulk of the lipids of all cellular membranes are phospholipids, the main precursor for all phospholipids is phosphatic acid (PA). PA is synthesized from glycerol-­‐3-­‐phosphate (G3P) by covalent binding of two acyl chains from Acyl-­‐CoA to the sn-­‐1 and sn-­‐2 positions of G3P. In mammals, the acetylation of G3P (to monoacyl G3P) occurs in the mitochondria and the acetylation of monoacyl G3P (to PA) occurs in the mitochondria and ER. In yeast the entire process occurs in the ER. PA can undergo phosphatase action to form diacylglycerol (DAG) or be coupled to cytidinediphosphate (CDP) to provide CDP-­‐DAG. Both DAG and CDP-­‐DAG are precursors for different lipids in separate pathways (Figure 1). CDP-­‐DAG provides Figure 1. Schematic overview of lipid metabolism in yeast and mammals. Conserved pathways are indicated by black arrows, yeast pathways are indicated by green arrows and m ammalian pathways are indicated by red arrows. Responsible genes are indicated in red for mammals and green for yeast. For detailed d escription of biochemical pathways and abbreviations, see text. the lipid backbone for phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin (CL). In both mammals and yeast the synthesis of PI takes place in the ER, whereas the synthesis of PG and CL take place in the mitochondria. In yeast, CDP-­‐
DAG is also the precursor for phosphatidylserine (PS). Phosphatidylethanolamine and phosphatidylcholine can be synthesized from DAG and CDP-­‐ethanolamine/CDP-­‐
choline, via a conserved pathway known as the Kennedy pathway (Figure 1, arrows from DAG to PE and PC). PS is decarboxylated to generate PE and PE is methylated to form PC in the Golgi and ER, respectively. In mammals however, the only pathway generating PS is via the carboxylation of PE in the ER. SPHINGOLIPIDS Sphingolipids and ceramide are an important component of the specialized membrane microdomains known as lipid rafts in the plasma membrane. Sphingolipids are typically elevated 50% in lipid rafts compared to the total PM. Synthesis of sphingolipids starts with Palmitoyl-­‐CoA, which is consumed in a condensation reaction with serine, to generate dehydrospringosine that is subsequently reduced to dihydriosphingosine. Dihydrosphingosine is oxidized to form sphingosine, which is the backbone for all sphingolipids. In mammals, ceramide is formed from sphingosine by the attachment of a (second) fatty acid to the free amine group and is the precursor for sphingomyelin and the complex glycopshingolipids. In yeast, dihydrosphingosine is hydroxylated to provide phytosphingosine, which is used to form phytoceramide. Phytoceramide is the precursor for α-­‐
hydroxyphytoceramide and inositol-­‐
phosphoceramides (IPC). Synthesis of ceramide or phytoceramide takes place in the ER, whereas complex sphingolipids are formed from ceramide in the Golgi. STEROLS Sterols are important for membrane fluidity and are found in all membranes, but the highest concentration of sterols is found in the plasma membrane. Acetyl-­‐
CoA is also the precursor for sterol synthesis. Acetyl-­‐CoA acetyl transferase proteins assemble acetoacetyl-­‐CoA out of two acetyl-­‐CoA molecules to form 3-­‐
hydroxy-­‐3-­‐methylglutaryl-­‐CoA (HMG-­‐CoA) that is then reduced and released from CoA to provide mevalonate. Mevalonate is the precursor for the synthesis of sterols in yeast (ergosterol) and mammals (cholesterol), which takes place in the ER. Steryl esters and other neutral lipids (ie. triglycerides) are stored in the lipid droplet (or adiposome). Lipid droplets consist of a core of mainly triglycerides and steryl esters, surrounded by a phospholipid monolayer and come into close approximation with the ER. The monolayer is coated by proteins that are involved in lipid metabolism and transport. Lipid droplets are mainly found in adipose (fat) tissue. Catabolism of lipids takes place via β-­‐
oxidation, which breaks down fatty acids into acetyl-­‐CoA. Fatty acids are oxidized mainly in the mitochondria. When fatty acids are to long for the mitochondria to process, they are broken down in the peroxisome. REGULATION OF LIPID BIOSYNTHESIS In yeast, the regulation of ergosterol and phospholipids are regulated by two different systems. Ergosterol synthesis is regulated by the sterol regulatory element (SRE) binding proteins (SREBPs). The SREBP Upc2p and its paralog Ecm22p are transcription factors that bind SREs promoters. Phospholipid synthesis is coordinated by an upstream activating sequence (UASIno), which is recognized by a heterodimeric transcription factor of Ino2p and Ino4p. Opi1p represses the Ino2p/Ino4p pathway by binding directly to Ino2p and recruiting a transcriptional repressor (Sin3p) to the promoter. Opi1p activity is, in turn, regulated by ER protein Scs2p and increased PA concentrations that block Opi1p export from the ER. In mammals, the synthesis of cholesterol and fatty acids is also regulated by the SREBP family of transcription factors. SREBPs are synthesized as inactive proteins, which become active after forming a complex with SREBP cleavage-­‐
activating protein (SCAP). The SCAP-­‐SREBP complex is incorporated in budding vesicles and transported from the ER to the Golgi where SCAP is cleaved by site-­‐1 and site-­‐2 proteases to release the active SREBP transcription factor into the cytosol for subsequent translocation into the nucleus. Membrane biogenesis, e.g. during phagocytosis, results in increased cleavage of SREBPs and increased transcription of lipogenic target genes, such as fatty acid synthase (FASN) and HMG-­‐CoA reductase (HMGCR). Cholesterol binds to SCAP and induces a conformational change that promotes interaction of SCAP with insulin induced gene (Insig) proteins and inhibits SCAP export from the ER as part of a negative feedback loop. Polyunsaturated saturated fatty acids (PUFAs) also decrease Insig turnover to suppress SREBP activation. THE UPR MECHANISM IN YEAST AND MAMMALS The mammalian UPR consists of three branches each represented by a key stress-­‐sensing protein, namely inositol requiring enzyme 1 (Ire1), the protein kinase-­‐like ER kinase (PERK) and the activating transcription factor 6 (ATF6). Each of the UPR-­‐sensors have lumenal stress-­‐sensing domains and cytoplasmic effector domains to communicate changes in the ER environment and coordinate highly integrated translational and transcriptional programs in order to maintain ER homeostasis (Brewer, 2014; Kimata & Kohno, 2011). IRE1 Ire1 is the most highly conserved branch of the UPR and is present from yeast to metazoan systems (Mori, 2009). Ire1 is named after the yeast homolog Ire1p (inositol requiring enzyme 1), which was first identified in an inositol auxotrophy screen (Nikawa & Yamashita, 1992) and was subsequently found to be required for cell viability under ER stress conditions (Cox, Shamu, & Walter, 1993). Mammals express two Ire1 isoforms, Ire1α and β: Ire1α is ubiquitously expressed, whereas Ire1β expression has only been reported in (gut) epithelial cells. Under normal conditions, Ire1 lumenal domain binds the ER chaperone BiP (Kar2p and Grp78p in yeast) that dissociates upon the induction of ER stress and the accumulation of unfolded proteins in the ER lumen (Bertolotti, Zhang, Hendershot, Harding, & Ron, 2000). However, BiP dissociation alone is not sufficient to induce Ire1 signaling and direct binding of unfolded proteins to the Ire1 lumenal domain is also required to induce oligomerisation and activate its cytoplasmic endoribonuclease (RNase) domain (Korennykh et al., 2009). The Ire1 RNase unconventionally cleaves XBP1 mRNA (HAC1 mRNA in yeast). Figure 2. Schematic overview of the UPR in yeast (a) and mammals (b-­‐d). (a) Yeast Ire1p splices HAC1 mRNA, which yields the active form of the Hac1p transcription factor. (b) Mammalian IRE1α splices XBP1 mRNA to yield the spliced mRNA (XBP1s), which is translated to the active XBP1 transcription factor. IRE1α also decays ER-­‐protein mRNA (regulated IRE1 dependent decay: RIDD). (c) PERK phosphorylates eIF2α, activating it as a transcription factor. (d) ATF6 is encapsulated and traffics into the Golgi, where it is spliced by site-­‐1/site-­‐2 directed proteases (S1P/S2P), releasing the active transcription factor from the membrane. Taken from (Kimata & Kohno, 2011). Spliced XBP1/HAC1 mRNA is then re-­‐
ligated to generate mRNA encoding the active bZiP transcription factor, XBP1 or Hac1p, that binds unfolded protein response elements (UPRE) in the nucleus to initiate transcription of various stress-­‐
alleviating genes involved in protein folding and lipid metabolism. However, the active Ire1 endonuclease also undertakes unconventional cleavage of many ER-­‐targeted mRNAs that are not subsequently re-­‐ligated and therefore targeted for degradation via a process called regulated IRE1-­‐dependent decay (RIDD) (Hollien et al., 2009). Via RIDD, the UPR can modulate the translation of mRNA and the flux of protein in the ER. PERK AND ATF6 Metazoans exhibit two additional UPR sensors, PERK and ATF6. The lumenal domain of PERK also dissociates from BiP during ER stress and, as it is highly homologous to the Ire1 lumenal domain, it is predicted to directly bind unfolded proteins. The PERK cytoplasmic domain is a serine/threonine kinase that dimerises and undergoes transautophosphorylation during ER stress. PERK is a member of the eIF2alpha kinase family and phosphorylates the α-­‐subunit of the eukaryotic initiation factor-­‐2 (eIF2) to inhibit global translation and reduce protein load on the ER. A number of mRNAs however are exempt from this inhibition including the transcription factor ATF4. ATF4 binds to CCAAT-­‐
enhancer binding protein-­‐activating transcription factor (C/EBP-­‐ATF) response elements (CARE) to induce transcription of UPR genes including those involved in amino-­‐acid synthesis and antioxidant responses. Downstream of ATF4, the CCAAT-­‐enhancer binding protein homology protein (CHOP) is also synthesized and, under prolonged stress, ATF4 and CHOP are thought to initiate apoptotic signalling events. The last of the three UPR branches is regulated by ATF6. Mammals express two isoforms of ATF6, namely ATF6α and ATF6β, however only ATF6alpha has been implicated in ER stress signaling. During stress, ATF6 dissociates from BiP and is transported to the Golgi via COPII coated vesicles where it undergoes proteolytic cleavage by site-­‐1 and -­‐2 proteases to free the ATF6 cytosolic domain (ATF6N). ATF6N is a bZiP transcription factor that translocates to the nucleus and binds ER stress responsive elements (ERSE) in the promoters of UPR-­‐responsive genes. Furthermore, ATF6N can heterodimerize with XBP1 to bind the UPR element (UPRE) with a high affinity and upregulate transcription of an alternative subset of genes suggesting that there are multiple modes, or phases, of UPR signaling. UPR ACTIVATION BY MEMBRANE STRESS The bulk of the membrane constitutes of lipids. The lipid classes and fatty acids determine the biochemical properties of the membrane such as membrane thickness, membrane fluidity and, together with specific proteins, membrane curvature. The thickness of the membrane is determined by the length and ordering of the acyl chains, providing the effective acyl chain length. Membrane fluidity is mainly determined by the lipid classes, acyl chains, sterol content and temperature. Together with curvature promoting proteins, the ratio between bilayer-­‐ and non-­‐bilayer preferring lipids (type I and II lipids respectively) determines the membrane curvature. Whether a lipid prefers formation bilayers depends on molecular shape, which is in turn determined by the acyl chains and the cross-­‐sectional area of the head group. These biophysical properties are essential for membrane and membrane-­‐protein function. All subcellular membranes have a specific membrane composition; for example, the endoplasmic reticulum has a membrane composition with the highest amount of PC, compared to the plasma membrane (PM) and other organelles (van Meer & de Kroon, 2011). In yeast, the width of the ER membrane is 7.5 ± 0.8 nm compared to 9.2 ± 0.4 for the PM. The ergosterol to PL ratio of the ER is 0.18 compared to 0.46 for the PM (Schneiter et al., 1999). To ensure proper function of the specific membranes, the metabolism of both lipid classes and acyl chains is tightly regulated. Perturbations in lipid homeostasis cause membrane stress and has been implicated in the activation of the UPR. It was shown that the transcription of genes for-­‐ and the activity of enzymes for PL synthesis is regulated by inositol in yeast (Greenberg & Lopes, 1996). When the concentration of inositol drops, genes involved in lipid synthesis (OPI3) and regulation of lipid synthesis (INO1, CHO1) are induced. Furthermore, the expression of the yeast BiP gene, KAR2, is four fold higher in inositol depleted medium (Cox, Chapman, & Walter, 1997), indicating a link between lipid metabolism and the UPR. The yeast knock out mutant of Δ-­‐
aminolevulinate (ALA) synthase (hem1Δ) is a key tool for investigating the relation between lipid metabolism and other cellular processes. The hem1Δ-­‐strain is incapable of forming heme – the prostetic group of the only yeast desaturase (Ole1p) and various enzymes involved in ergosterol biosynthesis. When transferred to an ALA-­‐deprived medium, ergosterol and UFAs are depleted from the membranes and the growth of the hem1Δ mutant arrests. Pineau et al. demonstrated that in ALA-­‐
deprived medium, the hem1Δ mutant could not deliver a GFP-­‐tagged membrane protein (the uracil permease Fur4p) to the plasma membrane and was rather diverted to the vacuolar lumen (Pineau et al., 2008). The inability of Fur4p to reach the plasma membrane was shown not to be due to an impaired secretory pathway. Supplementation of the medium with ergosterol or the UFA oleate (C18:1) could rescue the growth phenotype. Surprisingly, depletion of ergosterol or UFAs solely (by supplementing the ALA deprived media with oleate or sterols respectively) showed the same effect as depletion of both ergosterol and UFAs and did not deliver Fur4p to the plasma membrane. Further investigation established that the depletion of UFAs increases the amount of SFAs in membrane phospholipids and altered the biophysical properties of the membrane important for membrane protein function. For example, supplementation of the medium with the monounsaturated FA myristoleic acid (C14:1) rescued the degradation of Fur4p in the hem1Δ mutant by formation of diunsaturated lipids (e.g. with two myristoleic acid tails). This strongly suggested that alteration of the membrane biophysical properties (by increasing unsaturation) was responsible for the defect in Fur4p delivery. Diunsaturated lipids have a preference for hexagonal phase (non-­‐bilayer) in model membranes, which implicates that the formation of non-­‐bilayer preferring lipids rescues the phenotype. Indeed, it was further demonstrated that the presence of type II (non bilayer preferring) lipids was the critical parameter for Fur4p targeting to the PM and that membrane properties are essential for protein function and delivery. The same research group subsequently demonstrated that low levels of UFAs in the hem1Δ-­‐mutant induced ER stress and activated the UPR in absence of defects in the secretory pathway (Pineau et al., 2009). This effect was comparable to the effect of dithiothreitol (DTT), which disrupts protein folding by reducing disulfide-­‐bonds. Interestingly, providing hem1Δ-­‐cells with exogenous ergosterol (only depleting UFAs) increased induction of the UPR compared to depletion of both ergosterol and UFAs. In agreement with previous observations, UPR induction was abolished in the ole1Δ-­‐strain by providing the cell with exogenous oleate (C18:1), to maintain membrane unsaturation. UPR induction was confirmed to be dependent on the Ire1p/Hac1p pathway as the growth arrest observed for the hem1Δ ire1Δ double mutant was not relieved by supplementation with ergosterol, whereas this was the case in the hem1Δ mutant, indicating that synthesis of UFAs is necessary for cell growth in ire1Δ cells. Moreover, the addition of a chemical chaperone (4-­‐phenylbutyrate), which is known to alleviate chemical UPR induction, prevented induction of ER stress in hem1Δ-­‐ and ole1Δ-­‐cells. This suggested that Ire1p is required for growth under SFA accumulation. Indeed, exposure to the exogenous SFA palmitate (0.6 % in the presence of 0.1 % oleate) was shown to significantly alter ER morphology herefore it was concluded that the decrease of UFAs in the hem1Δ model induced the UPR by altering the biochemical properties of the ER membrane. Deguil et al. (2011) also used the hem1Δ model system to screen for fatty acids that alleviate ER stress and restore growth of the strain on ALA depletion and alleviation of UPR. Supplementation of the medium with various unsaturated fatty acids (UFAs) restored growth and out of the UFAs that restored growth of the hem1Δ cells, oleate showed the strongest rescuing effect. Contrary to the UFAs, none of the tested SFAs (even and odd numbered acyl chains, C10:0 -­‐ C24:0) could restore hem1Δ in ALA deprived medium. Moreover, in agreement with Pineau et al., supplementation with the SFA palmitate (C16:0) was shown to induce the UPR. To investigate whether the molecular shape of the fatty acid influences the capacity to restore growth of the hem1Δ cells, Deguil et al. used the MUFA oleate and the PUFAs linoleic acid (C18:2), linolenic acid (C18:3). At a concentration of 0.1 mM, oleate showed to be most effective in restoring growth and this effect was increased by higher concentrations (1 mM, 10 mM) of the FA. However, this effect was not observed for linoleic acid and linolenic acid exhibited toxicity at concentrations greater than 10 mM). Yhe observed rescuing effects were confirmed to be due to the incorporation of the monounsatutrated FAs into the membrane PC. From these studies it was clear that membrane perturbations induced ER stress and UPR in a highly specific manner, although the molecular mechanism was yet to be elucidated. A recent study by Promlek et al. (2011) has reported that unfolded proteins and membrane aberrancy have different mechanisms of UPR induction via Ire1. A truncation mutant of Ire1 named ΔIII (truncated in the lumenal stress sensing region) was compared to wild type Ire1. The ΔIII mutant was shown to have a later UPR onset in response to DTT or tunicamycin but comparable onset in response to membrane stress induced by inositol depletion. Furthermore, deletion of genes involved in secretory protein processing (eg. SCJ1, SPC2) or glycosylation (eg. ALG3, EOS1, PMT2) was shown to have milder induction of the UPR in the ΔIII-­‐mutant strain compared to WT Ire1 whereas such differences were not observed upon deletion of lipid metabolism genes (OPI3, SCS3, ISC1, MGA2). Promlek et al. therefore concluded that a different mechanism was responsible for the activation of Ire1 upon inositol depletion.This mechanism was further elucidated in mammalian cells by Volmer et.al. (2013) using murine cells lacking Ire1alpha (IRE1α -­‐/-­‐) and an IRE1α mutant (ΔLD-­‐IRE1α) lacking the lumenal domain. They found that pharmacological inhibition of the stearoyl-­‐CoA desaturase 1 (SCD1) induced UPR in the WT IRE1α strain as well as in the ΔLD-­‐IRE1α whereas incubation with tunicamycin and DTT only induced UPR in WT IRE1α cells. Comparing WT-­‐PERK to ΔLD-­‐PERK gave similar observations. This indicated that the membrane-­‐spanning and lumenal domain directly detects alterations in the ER membrane to induce downstream UPR signaling. Exposure to excess free fatty acids (FA), or lipotoxicity, is typical of metabolic diseases such as obesity and type II diabetes and ER-­‐stress has been observed in models in obese liver and adipose tissues (Ozcan et al., 2004)., Karaskov et.al. investigated whether FFAs could induce the UPR and demonstrated that exposure to saturated fatty acids (SFAs) induced PERK phosphorylation and eventually cell death in pancreatic β-­‐cells, a known cause of type II diabetes (Karaskov et al., 2006). Furthermore, apoptosis of mouse macrophages induced by lipotoxicity was shown to require a functional UPR-­‐pathway. (Devries-­‐Seimon et al., 2005). UPR INDUCED LIPID METABOLISM As outlined above, lipid imbalance or altered biochemical and biophysical properties of the membrane can cause ER stress and induce the UPR. However, UPR is also required to regulate lipid metabolism For example, early studies by Cox et.al. (1997) using multiple yeast knock out strains, demonstrated that the activation of Ire1p/ Hac1p is required for stimulating the expression of canonical UPRE-­‐regulated proteins but also the transcription of key genes required for phospholipid biosynthesis . Subsequent studies in mammalian systems, demonstrated that overexpression of the spliced XBP1 induced ER expansion by increasing the amount of PC per milligram of protein and the total amount of phospholipids (Sriburi, Jackowski, Mori, & Brewer, 2004). This appeared to be due to the increased activity of the choline phosphotransferases, CEPT1 and CMPT1, which synthesizes PC from CDP-­‐choline and DAG. XBP1 was also shown to regulate lipid synthesis via the acetyl-­‐CoA carbocylase ACACA, the DAG acyltransferase DGAT and the stearyl-­‐CoA decarboxylase SCD1 (Lee, Scapa, Cohen, & Glimcher, 2008). In XBP1Δ-­‐mice, ACACA, DGAT and SCD1 were shown to be downregulated but SREBP-­‐regulated genes were expressed at a normal level. No abnormalities in liver function or ER structure were observed, although the abundance of ER in the liver was decreased. These findings implicate XBP1 to have a role in regulation of lipid metabolism in a SREBP-­‐independent manner. ATF6αΔ mice were shown to accumulate lipid droplets in the liver upon chemical induction of ER stress although the deletion of the ATF6α gene had little effect on lipogenic gene expression (Rutkowski et al., 2008). However, Rutkowski et.al. observed that regulators of hepatic metabolism such as SREBP1 and the carbohydrate responsive element binding protein (ChREBP) were downregulated in both WT and knock-­‐out mice. The peroxisome proliferator-­‐
activated receptor α (PPARα) was also decreased and to a significantly greater extent in the ATF6α-­‐Δ mice. Interestingly, ATF6αΔ failed to upregulate transcription of these genes 48 hours after chemical induction of the UPR, indicating ongoing ER stress. However, perturbation of the ATF6-­‐branch of the UPR was shown to alter levels of XBP1-­‐mRNA splicing and it was concluded that ATF6 and XBP1 co-­‐
regulate lipid metabolism and that the accumulation of lipids in lipid droplets was possibly due to decreased fatty acid oxidation. In support of a role for ATF6 in regulating lipid metabolism, a constitutively active mutant of ATF6α, named ATF6α(1-­‐137), was shown to be able to enlarge the ER in a XBP1Δ mice fibroblast cell line (Bommiasamy et al., 2009). The ATF6α(1-­‐137) mutant was shown to increase the levels of the major membrane lipids PE and PC by 50-­‐60%. Activation via ATF6α(1-­‐137) increased fatty acid synthesis 3 – 3.5 fold and PC synthesis four-­‐fold due to a fourfold increase in CMPT and CEPT as previously observed for spliced XBP1 (Sriburi et al., 2004). In yeast it was shown that the transcription factor complex Ino2p/Ino4p involved in phospholipid biosynthesis is required for stress induced ER-­‐expansion (Schuck, Prinz, Thorn, Voss, & Walter, 2009). A constitutively active mutant of Ino2p, Ino2p(L119A), showed expansion of the ER without induction of ER stress. This expansion of the ER did not affect ER chaperones such as Kar2p and Pdi1p, which are up regulated by the canonical UPR. However, the ino2-­‐deletion mutant, that is incompetent for ER expansion, exhibited elevated amounts of ER chaperones when grown in the absence of exogenous lipids. Taken together, these results indicate that regulators of the UPR and lipid metabolism are highly integrated to maintain ER membrane homeostasis. Indeed, Thibault et. al. recently reported that activation of the UPR is required to maintain membrane morphology (Thibault et al., 2012). This study compared wild type cells to cells lacking the gene for phospholipid N-­‐
methyltransferase OPI3 and, using temperature-­‐sensitive regulation of IRE1 gene expression, revealed severe alterations in the membranes upon the inducible knock-­‐down of Ire1p. Furthermore, Thibault et al demonstrated that the expression of a membrane curvature inducing reticulon (Rtn2p) and the membrane stabilizing protein Hsp12p were increased upon UPR activation by lipid disequilibrium clearly indicating a critical role for Ire1p and the UPR in maintaining membrane structure. CONCLUDING REMARKS Here we have reviewed key recent literature describing a strong intersection between lipid metabolism and the UPR in maintaining ER homeostasis under both normal growth and (ER) stress conditions. For example, it was demonstrated that disruptions in the lipid homeostasis, represented by both changes in exogenous and endogenous level of fatty acids, could alter membrane composition and/or structure to induce ER stress in a highly specific manner. Further mechanistic studies have delineated that the membrane-­‐spanning domain of Ire1 detects perturbations in the ER membrane to initiate downstream UPR signaling, linking aberrancies in lipid metabolism directly to UPR induction. A number of recent studies have also implicated the UPR transcription factors, XBP1 and ATF6, in phospholipid homeostasis and ER morphology. This shows that the UPR does not only relieve ER stress by increasing protein folding capacity, but also by altering membrane homestasis. However, exactly how UPR-­‐
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Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains. Proc Natl Acad Sci U S A, 110(12), 4628-­‐4633. doi: 10.1073/pnas.1217611110 SUPPLEMENT: LAYMAN’S SUMMARY The endoplasmic reticulum (ER) is a cellular compartment that plays a central role in protein synthesis and is the main site for the synthesis of lipids, which are the main building blocks of all cellular membranes. When proteins are no longer folded correctly, they cause ER stress. ER-­‐stress sensors evoke a protective response, named the unfolded protein response (UPR). The UPR diminishes ER stress e.g. by increasing its capacity to synthesize and correctly fold proteins. The lipids in the membrane determine its biochemical and biophysical properties which are important for proper function of the membrane and membrane proteins and are therefore carefully regulated (de Kroon et al., 2013). The model eukaryote Saccharomyces cerevisiae (budding yeast or yeast in short) is a widely used system for research on ER-­‐ and lipid metabolism related subjects. In this review, we discussed the relation between ER-­‐stress by alterations in eukaryotic ER membrane composition in both yeast and mammalian model systems. ACTIVATION OF THE UPR BY M EMBRANE STRESS A yeast mutant has been described that accumulates saturated fatty acids at the expense of unsaturated fatty acids, thus altering the chemical properties of its lipids and membranes. This decrease of unsaturated fatty acids was shown to influence the delivery of a transport protein to the plasma membrane due to altered membrane composition in absence of defects in the secretory pathway (Pineau et al., 2008). However, low levels of unsaturated fatty acids still induced ER-­‐stress and activated the UPR in this mutant (Pineau et al., 2009). The addition of unsaturated fatty acids to the growth medium also alleviated ER-­‐stress (Deguil et al., 2011), indicating a link between UPR activation and altered membrane characteristics. The molecular mechanism of UPR activation by altered membrane characteristics was elucidated by deleting the unfolded protein-­‐sensing domain of a key UPR-­‐activating protein. This mutant was shown to be able to induce the UPR under ER-­‐stress caused by decreased amounts of unsaturated fatty acids, yet not by misfolded proteins (Volmer et al., 2013). Therefore it was concluded that the UPR can sense perturbations in membrane properties independent of its role in the classical unfolded protein response. UPR INDUCED LIPID M ETABOLISM The regulation of lipid synthesis is needed for expansion of the ER upon ER-­‐stress and, inversely, that expansion of the ER membrane alleviates ER stress (Schuck et al., 2009). UPR activation has been reported to increase the production of phospholipids and particularly the main membrane lipid, phosphatidylcholine (Sriburi et al., 2004). Moreover, the UPR appears to regulate membrane biosynthesis as activation of the UPR is needed to maintain membrane morphology upon knock out of one of the phosphatidylcholine synthesis genes (Thibault et al., 2012). CONCLUDING REMARKS The UPR is important in response to stress caused by the accumulation of unfolded proteins in the ER but can also to be activated by membrane stress or lipotoxicity. The UPR therefore resolves ER-­‐stress not only by improving protein synthesis, but also by regulating lipid metabolism. Although many mechanistic properties of the interplay between ER-­‐stress and lipid metabolism remain elusive,there is now evidence that there is an important link between these pathways that may prove important for therapeutic interventions in metabolic diseases