Endoplasmic Reticulum Stress and Lipid Metabolism 1 Mike F

ENDOPLASMIC RETICULUM STRESS AND LIPID METABOLISM 1 MIKE F. RENNE

ABSTRACT specialized for different functions and Upon endoplasmic reticulum (ER) stress, therefore differ in form. The cisternal ER the unfolded protein response (UPR) is continuous with the nuclear envelope triggers cellular mechanisms to restore and is studded with ribosomes, thus the ER homeostasis. Aberrancies in lipid cisternal sheets are the location of protein homeostasis can cause alterations in synthesis and folding. The tubular ER is biochemical and biophysical properties enriched in tissues specializing in the of the ER membrane, which impair ER biosynthesis of lipids and steroids and the function and induce UPR signaling. The large amount of enzymes required for UPR is also involved in regulation of lipid these processes. In addition to metabolism and membrane biogenesis, anterograde or retrograde vesicular indicating a link between ER-stress and transport, the ER can form membrane lipid homeostasis. In this review, we will junctions, or contact sites, with other discuss how alterations in lipid organelles to facilitate inter-organelle metabolism can activate the UPR, as well trafficking of lipids, enzymes and other as how the UPR alters lipid metabolism compounds. to relieve stress from the ER. When something goes wrong in cellular homeostasis, such as the accumulation of INTRODUCTION unfolded or misfolded proteins due to The endoplasmic reticulum is part of the inherited mutations, calcium- or oxidative endomembrane system, which further flux, the cell can be under (ER) stress. In consists of the nuclear envelope, plasma 1988, a stress response to unfolded membrane (PM), Golgi apparatus, proteins was reported, which we now endosomes, lysosomes, lipid droplets, know as the unfolded protein response peroxisomes and secretory vesicles. The (UPR) (Kozutsumi, Segal, Normington, ER is the entry point to the secretory Gething, & Sambrook, 1988). ER stress is pathway, and plays a central role in the synonymous with the activation of the synthesis of proteins but is also the site of UPR. The key signaling proteins for the phospholipid biosynthesis. It is UPR are ER-localised transmembrane responsible for all de novo synthesis of proteins that communicate perturbations membranes within the cell and has a within the lumen to initiate downstream dynamic membrane structure that allows cytoplasmic signaling cascades that fast expansion to accommodate protein function to resolve stress or, when in and membrane biosynthesis. The ER crisis, signal apoptosis. Chronic ER stress consists of the cisternal ER (historically has therefore been implicated in the referred to as “rough ER”) and tubular ER pathogenesis of many degenerative (historically referred to as “smooth ER”). diseases associated with lipid metabolism, Both compartments of the ER are

1Membrane Biochemistry & Biophysics, Bijvoet Centre for Biomolecular Research and Institute of Biomembranes, Utrecht University, the Netherlands including metabolic diseases (e.g. proper membrane (and membrane diabetes, obesity) and inflammatory protein) function, such as surface charge, diseases (e.g. artherosclerosis). While thickness and fluidity of the membrane cytosolic enzymes execute the synthesis (reviewed by de Kroon, Rijken, and De of fatty acids, the elongation of fatty acids Smet (2013)). De novo synthesis of lipids and the assembly of lipids occur starts with the formation of acetyl- predominantly in the ER. coenzyme A (Acetyl-CoA) in the cytosol. To synthesize of fatty acids, acetyl-CoA is This review will discuss studies from both carboxylated to malonyl-CoA. Fatty acid mammalian systems and the model synthases synthesize (mainly) palmitoyl- eukaryote Saccharomyces cerevisiae CoA from malonyl-CoA and acetyl-CoA; (baker’s yeast or yeast in short). Yeast is a palmitoyl-CoA can be processed widely used model eukoryote in studies (elongation, desaturation) in the ER to on ER-related subjects, such as ER-stress provide different fatty acids bound to CoA and lipid metabolism. Lipid metabolism (Acyl-CoA) (Figure 1). and regulation are highly conserved between yeast and mammals (Nielsen, In the following section we will briefly 2009). Furthermore, the yeast UPR is discuss the synthesis of lipids in mammals conserved in one of the branches of the and yeast. For a more complete coverage mammalian UPR, as well as one of the of these topics, the reader is referred to mammalian UPR branch is highly recent reviews (Henry, Kohlwein, & homologues to the yeast UPR (Mori, Carman, 2012; Holthuis & Menon, 2014; 2009). Nohturfft & Zhang, 2009) and references therein. There is accumulating evidence, from yeast and mammalian model systems, PHOSPHOLIPIDS that loss of lipid homeostasis causes ER The bulk of the lipids of all cellular stress and that the UPR is an important membranes are phospholipids, the main sensor of changes in ER membrane precursor for all phospholipids is homeostasis and is required for lipid phosphatic acid (PA). PA is synthesized biosynthesis. Therefore, this review will from glycerol-3-phosphate (G3P) by discuss the intersection between the UPR covalent binding of two acyl chains from and lipid metabolism. Acyl-CoA to the sn-1 and sn-2 positions of G3P. In mammals, the acetylation of G3P LIPID METABOLISM AT A GLANCE (to monoacyl G3P) occurs in the Membrane lipids can be discriminated on mitochondria and the acetylation of the base of their chemical structure, monoacyl G3P (to PA) occurs in the dividing them into three main families: mitochondria and ER. In yeast the entire glycerophospholipids (phospholipids, PL), process occurs in the ER. PA can undergo glycerosphingolipids (sphingolipids, SL) phosphatase action to form diacylglycerol and sterols. Within these three families, (DAG) or be coupled to there are various different classes in cytidinediphosphate (CDP) to provide which thousands of different molecular CDP-DAG. Both DAG and CDP-DAG are species have been identified. The lipid precursors for different lipids in separate composition of the membrane determines pathways (Figure 1). CDP-DAG provides the biophysical properties essential for

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 mammalian pathways are indicated by red arrows. Responsible genes are indicated in red for mammals and green for yeast. For detailed description of biochemical pathways and abbreviations, see text.

the lipid backbone for rafts in the plasma membrane. phosphatidylinositol (PI), Sphingolipids are typically elevated 50% in phosphatidylglycerol (PG) and cardiolipin lipid rafts compared to the total PM. (CL). In both mammals and yeast the Synthesis of sphingolipids starts with synthesis of PI takes place in the ER, Palmitoyl-CoA, which is consumed in a whereas the synthesis of PG and CL take condensation reaction with serine, to place in the mitochondria. In yeast, CDP- generate dehydrospringosine that is DAG is also the precursor for subsequently reduced to phosphatidylserine (PS). dihydriosphingosine. Dihydrosphingosine Phosphatidylethanolamine and is oxidized to form sphingosine, which is phosphatidylcholine can be synthesized the backbone for all sphingolipids. In from DAG and CDP-ethanolamine/CDP- mammals, ceramide is formed from choline, via a conserved pathway known sphingosine by the attachment of a as the Kennedy pathway (Figure 1, arrows (second) fatty acid to the free amine from DAG to PE and PC). PS is group and is the precursor for decarboxylated to generate PE and PE is sphingomyelin and the complex methylated to form PC in the Golgi and glycopshingolipids. In yeast, ER, respectively. In mammals however, dihydrosphingosine is hydroxylated to the only pathway generating PS is via the provide phytosphingosine, which is used carboxylation of PE in the ER. to form phytoceramide. Phytoceramide is the precursor for α- SPHINGOLIPIDS hydroxyphytoceramide and inositol- Sphingolipids and ceramide are an phosphoceramides (IPC). Synthesis of important component of the specialized ceramide or phytoceramide takes place in membrane microdomains known as lipid the ER, whereas complex sphingolipids are transcription factors that bind SREs are formed from ceramide in the Golgi. promoters. Phospholipid synthesis is coordinated by an upstream activating STEROLS sequence (UASIno), which is recognized by Sterols are important for membrane a heterodimeric transcription factor of fluidity and are found in all membranes, Ino2p and Ino4p. Opi1p represses the but the highest concentration of sterols is Ino2p/Ino4p pathway by binding directly found in the plasma membrane. Acetyl- to Ino2p and recruiting a transcriptional CoA is also the precursor for sterol repressor (Sin3p) to the promoter. Opi1p synthesis. Acetyl-CoA acetyl transferase activity is, in turn, regulated by ER protein proteins assemble acetoacetyl-CoA out of Scs2p and increased PA concentrations two acetyl-CoA molecules to form 3- that block Opi1p export from the ER. hydroxy-3-methylglutaryl-CoA (HMG-CoA) that is then reduced and released from In mammals, the synthesis of cholesterol CoA to provide mevalonate. Mevalonate and fatty acids is also regulated by the is the precursor for the synthesis of SREBP family of transcription factors. sterols in yeast (ergosterol) and mammals SREBPs are synthesized as inactive (cholesterol), which takes place in the ER. proteins, which become active after forming a complex with SREBP cleavage- Steryl esters and other neutral lipids (ie. activating protein (SCAP). The SCAP-SREBP triglycerides) are stored in the lipid complex is incorporated in budding droplet (or adiposome). Lipid droplets vesicles and transported from the ER to consist of a core of mainly triglycerides the Golgi where SCAP is cleaved by site-1 and steryl esters, surrounded by a and site-2 proteases to release the active phospholipid monolayer and come into SREBP transcription factor into the cytosol close approximation with the ER. The for subsequent translocation into the monolayer is coated by proteins that are nucleus. Membrane biogenesis, e.g. involved in lipid metabolism and during phagocytosis, results in increased transport. Lipid droplets are mainly found cleavage of SREBPs and increased in adipose (fat) tissue. transcription of lipogenic target genes, such as fatty acid synthase (FASN) and Catabolism of lipids takes place via β- HMG-CoA reductase (HMGCR). oxidation, which breaks down fatty Cholesterol binds to SCAP and induces a acids into acetyl-CoA. Fatty acids are oxidized mainly in the mitochondria. conformational change that promotes When fatty acids are to long for the interaction of SCAP with insulin induced mitochondria to process, they are gene (Insig) proteins and inhibits SCAP broken down in the peroxisome. export from the ER as part of a negative feedback loop. Polyunsaturated saturated REGULATION OF LIPID BIOSYNTHESIS fatty acids (PUFAs) also decrease Insig In yeast, the regulation of ergosterol and turnover to suppress SREBP activation. phospholipids are regulated by two different systems. Ergosterol synthesis is THE UPR MECHANISM IN YEAST AND regulated by the sterol regulatory MAMMALS element (SRE) binding proteins (SREBPs). The mammalian UPR consists of three The SREBP Upc2p and its paralog Ecm22p 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 Figure 2. Schematic overview of the UPR in first identified in an inositol auxotrophy yeast (a) and mammals (b-d). (a) Yeast Ire1p screen (Nikawa & Yamashita, 1992) and splices HAC1 mRNA, which yields the active was subsequently found to be required form of the Hac1p transcription factor. (b) for cell viability under ER stress conditions Mammalian IRE1α splices XBP1 mRNA to (Cox, Shamu, & Walter, 1993). Mammals yield the spliced mRNA (XBP1s), which is express two Ire1 isoforms, Ire1α and β: translated to the active XBP1 transcription Ire1α is ubiquitously expressed, whereas factor. IRE1α also decays ER-protein mRNA Ire1β expression has only been reported (regulated IRE1 dependent decay: RIDD). (c) in (gut) epithelial cells. PERK phosphorylates eIF2α, activating it as a transcription factor. (d) ATF6 is Under normal conditions, Ire1 lumenal encapsulated and traffics into the Golgi, domain binds the ER chaperone BiP where it is spliced by site-1/site-2 directed (Kar2p and Grp78p in yeast) that proteases (S1P/S2P), releasing the active dissociates upon the induction of ER transcription factor from the membrane. stress and the accumulation of unfolded Taken from (Kimata & Kohno, 2011). proteins in the ER lumen (Bertolotti, Zhang, Hendershot, Harding, & Ron, Spliced XBP1/HAC1 mRNA is then re- 2000). However, BiP dissociation alone is ligated to generate mRNA encoding the not sufficient to induce Ire1 signaling and active bZiP transcription factor, XBP1 or direct binding of unfolded proteins to the Hac1p, that binds unfolded protein Ire1 lumenal domain is also required to response elements (UPRE) in the nucleus induce oligomerisation and activate its to initiate transcription of various stress- cytoplasmic endoribonuclease (RNase) alleviating genes involved in protein domain (Korennykh et al., 2009). folding and lipid metabolism. However, The Ire1 RNase unconventionally cleaves the active Ire1 endonuclease also XBP1 mRNA (HAC1 mRNA in yeast). undertakes unconventional cleavage of many ER-targeted mRNAs that are not coated vesicles where it undergoes subsequently re-ligated and therefore proteolytic cleavage by site-1 and -2 targeted for degradation via a process proteases to free the ATF6 cytosolic called regulated IRE1-dependent decay domain (ATF6N). ATF6N is a bZiP (RIDD) (Hollien et al., 2009). Via RIDD, the transcription factor that translocates to UPR can modulate the translation of the nucleus and binds ER stress mRNA and the flux of protein in the ER. responsive elements (ERSE) in the promoters of UPR-responsive genes. PERK AND ATF6 Furthermore, ATF6N can heterodimerize Metazoans exhibit two additional UPR with XBP1 to bind the UPR element sensors, PERK and ATF6. The lumenal (UPRE) with a high affinity and upregulate domain of PERK also dissociates from BiP transcription of an alternative subset of during ER stress and, as it is highly genes suggesting that there are multiple homologous to the Ire1 lumenal domain, modes, or phases, of UPR signaling. it is predicted to directly bind unfolded proteins. The PERK cytoplasmic domain is UPR ACTIVATION BY MEMBRANE STRESS a serine/threonine kinase that dimerises The bulk of the membrane constitutes of and undergoes transautophosphorylation lipids. The lipid classes and fatty acids during ER stress. PERK is a member of the determine the biochemical properties of eIF2alpha kinase family and the membrane such as membrane phosphorylates the α-subunit of the thickness, membrane fluidity and, eukaryotic initiation factor-2 (eIF2) to together with specific proteins, inhibit global translation and reduce membrane curvature. The thickness of protein load on the ER. A number of the membrane is determined by the mRNAs however are exempt from this length and ordering of the acyl chains, inhibition including the transcription providing the effective acyl chain length. factor ATF4. ATF4 binds to CCAAT- Membrane fluidity is mainly determined enhancer binding protein-activating by the lipid classes, acyl chains, sterol transcription factor (C/EBP-ATF) content and temperature. Together with response elements (CARE) to induce curvature promoting proteins, the ratio transcription of UPR genes including between bilayer- and non-bilayer those involved in amino-acid synthesis preferring lipids (type I and II lipids and antioxidant responses. Downstream respectively) determines the membrane of ATF4, the CCAAT-enhancer binding curvature. Whether a lipid prefers protein homology protein (CHOP) is formation bilayers depends on molecular also synthesized and, under prolonged shape, which is in turn determined by the stress, ATF4 and CHOP are thought to acyl chains and the cross-sectional area of initiate apoptotic signalling events. the head group. These biophysical The last of the three UPR branches is properties are essential for membrane regulated by ATF6. Mammals express two and membrane-protein function. All isoforms of ATF6, namely ATF6α and subcellular membranes have a specific ATF6β, however only ATF6alpha has membrane composition; for example, the been implicated in ER stress signaling. endoplasmic reticulum has a membrane During stress, ATF6 dissociates from BiP composition with the highest amount of and is transported to the Golgi via COPII PC, compared to the plasma membrane (PM) and other organelles (van Meer & de al., 2008). The inability of Fur4p to reach Kroon, 2011). In yeast, the width of the ER the plasma membrane was shown not to membrane is 7.5 ± 0.8 nm compared to be due to an impaired secretory pathway. 9.2 ± 0.4 for the PM. The ergosterol to PL Supplementation of the medium with ratio of the ER is 0.18 compared to 0.46 ergosterol or the UFA oleate (C18:1) could for the PM (Schneiter et al., 1999). To rescue the growth phenotype. ensure proper function of the specific Surprisingly, depletion of ergosterol or membranes, the metabolism of both lipid UFAs solely (by supplementing the ALA classes and acyl chains is tightly regulated. deprived media with oleate or sterols respectively) showed the same effect as Perturbations in lipid homeostasis cause depletion of both ergosterol and UFAs membrane stress and has been implicated and did not deliver Fur4p to the plasma in the activation of the UPR. It was shown membrane. Further investigation that the transcription of genes for- and established that the depletion of UFAs the activity of enzymes for PL synthesis is increases the amount of SFAs in regulated by inositol in yeast (Greenberg membrane phospholipids and altered the & Lopes, 1996). When the concentration biophysical properties of the membrane of inositol drops, genes involved in lipid important for membrane protein synthesis (OPI3) and regulation of lipid function. For example, supplementation synthesis (INO1, CHO1) are induced. of the medium with the monounsaturated Furthermore, the expression of the yeast FA myristoleic acid (C14:1) rescued the BiP gene, KAR2, is four fold higher in degradation of Fur4p in the hem1Δ inositol depleted medium (Cox, Chapman, mutant by formation of diunsaturated & Walter, 1997), indicating a link between lipids (e.g. with two myristoleic acid tails). lipid metabolism and the UPR. This strongly suggested that alteration of The yeast knock out mutant of Δ- the membrane biophysical properties (by aminolevulinate (ALA) synthase (hem1Δ) increasing unsaturation) was responsible is a key tool for investigating the relation for the defect in Fur4p delivery. between lipid metabolism and other Diunsaturated lipids have a preference for cellular processes. The hem1Δ-strain is hexagonal phase (non-bilayer) in model incapable of forming heme – the prostetic membranes, which implicates that the group of the only yeast desaturase formation of non-bilayer preferring lipids (Ole1p) and various enzymes involved in rescues the phenotype. Indeed, it was ergosterol biosynthesis. When transferred further demonstrated that the presence to an ALA-deprived medium, ergosterol of type II (non bilayer preferring) lipids and UFAs are depleted from the was the critical parameter for Fur4p membranes and the growth of the hem1Δ targeting to the PM and that membrane mutant arrests. properties are essential for protein function and delivery. Pineau et al. demonstrated that in ALA- deprived medium, the hem1Δ mutant The same research group subsequently could not deliver a GFP-tagged membrane demonstrated that low levels of UFAs in protein (the uracil permease Fur4p) to the the hem1Δ-mutant induced ER stress and plasma membrane and was rather activated the UPR in absence of defects in diverted to the vacuolar lumen (Pineau et the secretory pathway (Pineau et al., 2009). This effect was comparable to the strongest rescuing effect. Contrary to the effect of dithiothreitol (DTT), which UFAs, none of the tested SFAs (even and disrupts protein folding by reducing odd numbered acyl chains, C10:0 - C24:0) disulfide-bonds. Interestingly, providing could restore hem1Δ in ALA deprived hem1Δ-cells with exogenous ergosterol medium. Moreover, in agreement with (only depleting UFAs) increased induction Pineau et al., supplementation with the of the UPR compared to depletion of both SFA palmitate (C16:0) was shown to ergosterol and UFAs. In agreement with induce the UPR. To investigate whether previous observations, UPR induction was the molecular shape of the fatty acid abolished in the ole1Δ-strain by providing influences the capacity to restore growth the cell with exogenous oleate (C18:1), to of the hem1Δ cells, Deguil et al. used the maintain membrane unsaturation. UPR MUFA oleate and the PUFAs linoleic acid induction was confirmed to be dependent (C18:2), linolenic acid (C18:3). At a on the Ire1p/Hac1p pathway as the concentration of 0.1 mM, oleate showed growth arrest observed for the hem1Δ to be most effective in restoring growth ire1Δ double mutant was not relieved by and this effect was increased by higher supplementation with ergosterol, concentrations (1 mM, 10 mM) of the FA. whereas this was the case in the hem1Δ However, this effect was not observed for mutant, indicating that synthesis of UFAs linoleic acid and linolenic acid exhibited is necessary for cell growth in ire1Δ cells. toxicity at concentrations greater than 10 Moreover, the addition of a chemical mM). Yhe observed rescuing effects were chaperone (4-phenylbutyrate), which is confirmed to be due to the incorporation known to alleviate chemical UPR of the monounsatutrated FAs into the induction, prevented induction of ER membrane PC. From these studies it was stress in hem1Δ- and ole1Δ-cells. This clear that membrane perturbations suggested that Ire1p is required for induced ER stress and UPR in a highly growth under SFA accumulation. Indeed, specific manner, although the molecular exposure to the exogenous SFA palmitate mechanism was yet to be elucidated. (0.6 % in the presence of 0.1 % oleate) A recent study by Promlek et al. (2011) was shown to significantly alter ER morphology herefore it was concluded has reported that unfolded proteins and membrane aberrancy have different that the decrease of UFAs in the hem1Δ mechanisms of UPR induction via Ire1. A model induced the UPR by altering the biochemical properties of the ER truncation mutant of Ire1 named ΔIII (truncated in the lumenal stress sensing membrane. region) was compared to wild type Ire1. Deguil et al. (2011) also used the hem1Δ The ΔIII mutant was shown to have a later model system to screen for fatty acids UPR onset in response to DTT or that alleviate ER stress and restore growth tunicamycin but comparable onset in of the strain on ALA depletion and response to membrane stress induced by alleviation of UPR. Supplementation of inositol depletion. Furthermore, deletion the medium with various unsaturated of genes involved in secretory protein fatty acids (UFAs) restored growth and processing (eg. SCJ1, SPC2) or out of the UFAs that restored growth of glycosylation (eg. ALG3, EOS1, PMT2) was the hem1Δ cells, oleate showed the shown to have milder induction of the UPR in the ΔIII-mutant strain compared to As outlined above, lipid imbalance or WT Ire1 whereas such differences were altered biochemical and biophysical not observed upon deletion of lipid properties of the membrane can cause ER metabolism genes (OPI3, SCS3, ISC1, stress and induce the UPR. However, UPR MGA2). Promlek et al. therefore is also required to regulate lipid concluded that a different mechanism metabolism For example, early studies by was responsible for the activation of Ire1 Cox et.al. (1997) using multiple yeast upon inositol depletion.This mechanism knock out strains, demonstrated that the was further elucidated in mammalian cells activation of Ire1p/ Hac1p is required for by Volmer et.al. (2013) using murine cells stimulating the expression of canonical lacking Ire1alpha (IRE1α -/-) and an IRE1α UPRE-regulated proteins but also the mutant (ΔLD-IRE1α) lacking the lumenal transcription of key genes required for domain. They found that pharmacological phospholipid biosynthesis . inhibition of the stearoyl-CoA desaturase Subsequent studies in mammalian 1 (SCD1) induced UPR in the WT IRE1α systems, demonstrated that strain as well as in the ΔLD-IRE1α whereas incubation with tunicamycin and DTT only overexpression of the spliced XBP1 induced ER expansion by increasing the induced UPR in WT IRE1α cells. amount of PC per milligram of protein and Comparing WT-PERK to ΔLD-PERK gave similar observations. This indicated that the total amount of phospholipids (Sriburi, Jackowski, Mori, & Brewer, the membrane-spanning and lumenal 2004). This appeared to be due to the domain directly detects alterations in the increased activity of the choline ER membrane to induce downstream UPR phosphotransferases, CEPT1 and CMPT1, signaling. which synthesizes PC from CDP-choline Exposure to excess free fatty acids (FA), or and DAG. XBP1 was also shown to lipotoxicity, is typical of metabolic regulate lipid synthesis via the acetyl-CoA diseases such as obesity and type II carbocylase ACACA, the DAG diabetes and ER-stress has been observed acyltransferase DGAT and the stearyl-CoA in models in obese liver and adipose decarboxylase SCD1 (Lee, Scapa, Cohen, & tissues (Ozcan et al., 2004)., Karaskov Glimcher, 2008). In XBP1Δ-mice, ACACA, et.al. investigated whether FFAs could DGAT and SCD1 were shown to be induce the UPR and demonstrated that downregulated but SREBP-regulated exposure to saturated fatty acids (SFAs) genes were expressed at a normal level. induced PERK phosphorylation and No abnormalities in liver function or ER eventually cell death in pancreatic β-cells, structure were observed, although the a known cause of type II diabetes abundance of ER in the liver was (Karaskov et al., 2006). Furthermore, decreased. These findings implicate XBP1 apoptosis of mouse macrophages induced to have a role in regulation of lipid by lipotoxicity was shown to require a metabolism in a SREBP-independent functional UPR-pathway. (Devries-Seimon manner. et al., 2005). ATF6αΔ mice were shown to accumulate UPR INDUCED LIPID METABOLISM lipid droplets in the liver upon chemical induction of ER stress although the deletion of the ATF6α gene had little of the ER without induction of ER stress. effect on lipogenic gene expression This expansion of the ER did not affect ER (Rutkowski et al., 2008). However, chaperones such as Kar2p and Pdi1p, Rutkowski et.al. observed that regulators which are up regulated by the canonical of hepatic metabolism such as SREBP1 UPR. However, the ino2-deletion mutant, and the carbohydrate responsive element that is incompetent for ER expansion, binding protein (ChREBP) were exhibited elevated amounts of ER downregulated in both WT and knock-out chaperones when grown in the absence of mice. The peroxisome proliferator- exogenous lipids. Taken together, these activated receptor α (PPARα) was also results indicate that regulators of the UPR decreased and to a significantly greater and lipid metabolism are highly integrated extent in the ATF6α-Δ mice. Interestingly, to maintain ER membrane homeostasis. ATF6αΔ failed to upregulate transcription Indeed, Thibault et. al. recently reported of these genes 48 hours after chemical that activation of the UPR is required to induction of the UPR, indicating ongoing maintain membrane morphology ER stress. However, perturbation of the (Thibault et al., 2012). This study ATF6-branch of the UPR was shown to compared wild type cells to cells lacking alter levels of XBP1-mRNA splicing and it the gene for phospholipid N- was concluded that ATF6 and XBP1 co- methyltransferase OPI3 and, using regulate lipid metabolism and that the temperature-sensitive regulation of IRE1 accumulation of lipids in lipid droplets gene expression, revealed severe was possibly due to decreased fatty acid alterations in the membranes upon the oxidation. In support of a role for ATF6 in inducible knock-down of Ire1p. regulating lipid metabolism, a Furthermore, Thibault et al demonstrated constitutively active mutant of ATF6α, that the expression of a membrane named ATF6α(1-137), was shown to be curvature inducing reticulon (Rtn2p) and able to enlarge the ER in a XBP1Δ mice the membrane stabilizing protein Hsp12p fibroblast cell line (Bommiasamy et al., were increased upon UPR activation by 2009). The ATF6α(1-137) mutant was lipid disequilibrium clearly indicating a shown to increase the levels of the critical role for Ire1p and the UPR in major membrane lipids PE and PC by maintaining membrane structure. 50-60%. Activation via ATF6α(1-137) increased fatty acid synthesis 3 – 3.5 CONCLUDING REMARKS fold and PC synthesis four-fold due to a Here we have reviewed key recent fourfold increase in CMPT and CEPT as literature describing a strong intersection previously observed for spliced XBP1 between lipid metabolism and the UPR in (Sriburi et al., 2004). maintaining ER homeostasis under both normal growth and (ER) stress conditions. In yeast it was shown that the For example, it was demonstrated that transcription factor complex Ino2p/Ino4p disruptions in the lipid homeostasis, involved in phospholipid biosynthesis is represented by both changes in required for stress induced ER-expansion exogenous and endogenous level of fatty (Schuck, Prinz, Thorn, Voss, & Walter, acids, could alter membrane composition 2009). A constitutively active mutant of and/or structure to induce ER stress in a Ino2p, Ino2p(L119A), showed expansion highly specific manner. Further mechanistic studies have delineated that regulation (ie. Ino2p/Ino4p in yeast) is the membrane-spanning domain of Ire1 currently unclear although these detects perturbations in the ER intersections appear to be governed by membrane to initiate downstream UPR highly dynamic, and complex regulatory signaling, linking aberrancies in lipid mechanisms. Chronic ER stress signaling metabolism directly to UPR induction. A and apoptosis has been implicated in the number of recent studies have also pathogenesis of many serious implicated the UPR transcription factors, degenerative diseases, including XBP1 and ATF6, in phospholipid metabolic diseases such as type II homeostasis and ER morphology. This diabetes. Therefore, understanding the shows that the UPR does not only relieve relationship between lipotoxcity and ER ER stress by increasing protein folding stress, and relevant feedback regulation, capacity, but also by altering membrane represents an important topic in homestasis. However, exactly how UPR- designing novel treatments of these induced transcriptional programs are diseases. integrated into classical lipid metabolism

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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 MEMBRANE 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 METABOLISM 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