Endoplasmicreticulum-induced signal transduction and gene expression

Endoplasmicreticulum-induced . *_ .a _a signal transduction and gene expression Cells can respond to perturbations

in endoplasmic

reticulum

function by activating two distinct signal-transduction

(RR)

pathways:

one responds to unfolded proteins, the other to an overload of the organelle with membraneproteins.

A third pathway is activated upon

sterol depletion of cells and involves the cleavage and subsequent nuclear banslocation of an ER membrane-bound Thus, three distinctpathways

transcription factor.

each activated by a different signal

are currently known to project from the ER into the nucleus. This review summarizes the current understanding of these three pathways.

Heike Pahl is at the Institute for Experimental Cancer Research, Tumor Biology Center, PO Box 1120, D-79106 Freiburg, Germany; and Patrick Baeuerle is at Tularik, Inc., Two Corporate Drive, South San Francisco, CA 94080, USA. E-mails: [email protected] baeuerle@tularik. rapids.com

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A variety of organelles in eukaryotic cells provide optimal environments for specific biochemical reactions. For example, the formation of disulphide bonds during folding of secretory proteins occurs under oxidizing conditions in the lumen of the endoplasmic reticulum (ER), which is separated by a membrane from the reducing environment of the cytosol. Because signalling molecules cannot diffuse freely between the exterior and interior of a cell or between the lumen of organelles and the cytosol, sophisticated signaltransduction pathways have evolved. While extensive knowledge has accumulated about signal-transduction pathways linking the cell surface and the transcrlption apparatus in the nucleus, very little is known about pathways allowing particular organelles to elicit a genomic response. Such pathways are very important as they allow the cell to adjust the protein composition and size of organelles to changing needs. For example, peroxisome proliferators such as cloflbrate and Wy-14,643 have been shown to activate a specific transcription factor, PPAR, capable of coordinately activating peroxisomal gene expression1,2. Another organelle that needs to sense functional perturbations and to respond by activating novel gene expression in the nucleus is the ER. The existence of such a signalling pathway was first reported by Kozutsumi et uL3, who showed that the expression of mutant but not of wild-type influenza haemagglutinin induced the expression of genes for several ER-resident proteins. These proteins, first named glucose-regulated proteins (GRPs) because of their induction by glucose starvation, include the heavy chain binding protein BiP/GRP78, GRP94, protein-dlsulphide isomerase Copyright

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Elsevier

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Ltd. All rights

resewed.

0962~8924/97/517.00

(PDI/ERp59) and ERp72. GRPs facilitate protein folding in the ER, thereby alleviating ER stress. Because the common stimulus for induction of GRPs is the presence of malfolded proteins in the ER (Ref. 3), this pathway was named the unfolded-protein response (UPR). Yeast cells contain proteins homologous to the mammalian GRPs and can also mount a UPR (Ref. 4). A second ER-nucleus pathway, which wasonly identified very recently, is activated by the accumulation of membrane proteins in the ER. This condition may be of pathophysiological significance upon viral infection when the organelle is overcharged by the production of viral membrane proteins or, in several genetic diseases, such as cystic fibrosis, when genetically altered proteins accumulate in the ER. Finally, the best-characterized paradigm of ER-nucleus signal transduction and gene expression is the transcription factor SREBP, the sterol regulatory element-binding protein. This ER-resident pro-transcription factor responds to alterations in the lipid composition of the ER membrane. Recent work suggests that SREBP is not only involved in regulating sterol synthesis but also in coordinating lipid metabolism. The unfolded-protein

response

in yeast

Because of the easewith which genetic experiments can be conducted in Saccharomyces cerevisiae, more components of the UPR have been identified in this system than in mammalian cells. Since it is not yet clear whether individual elements of the pathways are evolutionarily conserved, we will discuss the yeast and the mammalian UPR separately. Treatment of yeast cells with a variety of agents that perturb ER function induces transcription of several ER-resident proteins4. Deletion analysis of the KAR2 promoter identified a 22-bp element, the unfolded-protein response element (UPRE), that is conserved in the promoters of all UPR target genes and is required for their induction by ER stresss. Moreover, the UPRE confers ER stress inducibility on a heterologous promoter. Recently, a novel transcription factor named Haclp (HAC: homologous to ATF and CREB) was identified, which binds to the UPRE during ER stress6 (see Fig. la). HACl is required for activation of the UPR since yeast strains deleted for this gene no longer induce the response. Haclp activity is regulated in an unusual and complex manner. One form of Haclp, Haclp”, is synthesized constitutively. However, upon induction of the UPR, the HACl mRNA is spliced at a nonconsensus splice site to generate an alternative protein product, Haclp’ (Ref. 6; see Fig. la). This alternative splicing requires tRNA ligase (RLGI) activity but not the spliceosome, which normally is responsible for mRNA processing7. A mutation in RLGl that abolishes splicing of the &Xl mRNA, and hence prevents induction of the UPR, has no effect on tRNA splicing, indicating that the tRNA ligase encodes two distinct enzymatic activities. Expression of Haclp’ constitutively activates the UPR, but heterologously expressed unspliced Haclp” only becomes active after the UPR is triggered. Haclp” contains a PEST sequence, a region often found in proteins targeted for trends in CELL BIOLOGY

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(4 f

(b)

Ca*+

Haclp’

q

.

mRNA

-I KB element

-

CytokinZQZY transcription

EUG 7

FIGURE

1

(a) The unfolded-protein response in yeast. When unfolded proteins accumulate in the ER, Ire1 p, a Serflhr kinase in the ER membrane and/or nuclear envelope, becomes activated by oligomerfzation and autophosphorylates. This leads to splicing of the HACl mRNA, resulting in the production of the Hacl pi protein. Ire1 p may possess exonuclease activity and participate in the splicing process. Hacl p enters the nucleus and binds to its cognate DNA sequence, the UPRE, leading to transcription of the KARL and fUGI genes. Their products aid protein folding in the ER, thereby alleviating the ER stress that initiated the unfolded-protein response. Abbreviation: UPRE, unfolded-protein response element. (b) The ER-overload response. An accumulation of proteins in the ER triggers the release of Ca*+ from the organelle by a yet-unknown mechanism. CaZ+ release leads to the production of ROls by the peroxidase moiety of lipoxygenases and cyclooxygenases. Increasing cellular concentrations of ROls cause the phosphorylation and subsequent degradation of IxB, the inhibitory subunit of NF-KB. This releases the active NF-rB heterodimer, which translocates to the nucleus and increases transcription of target genes. Abbreviations: ROls, reactive oxygen intermediates. (c) The SREBP pathway. During sterol SREBP depletion of cells, the SREBP precursor protein, which is inserted into the ER membrane, is cleaved by two proteases. The first protease resides in the ER and cleaves the protein in its ER ‘lumenal loop’, the second protease is ER membrane bound and cleaves on the cytoplasmic side of the membrane. Cleavage releases a SOO-amino-acid fragment that upon dimerization translocates to the nucleus. There it binds the sterol regulatory element, increasing transcription of genes involved in cholesterol uptake from the serum and in cholesterol synthesis as well as fatty acid and lipid synthesis. Abbreviations: ACC, acetyl coenzyme A carboxylase; FAS, fatty acid synthase; FPP, famesyl diphosphate synthase; HMCS, 3-hydroxy-3-methylglutaryl CoA synthase; LDL-R, low-density lipoprotein receptor; SRE, sterol regulatory element; SREBP, sterol regulatory-element binding protein.

rapid degradation by the proteasome, and appears to be much more rapidly degraded than Haclp’. When expressed in yeast strains that are incapable of degrading the protein, HaclpU functions as a transcription factor. Rapid degradation of Haclp” is likely, therefore, to be the reason for the lack of trends in CELL BIOLOGY

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transcriptional response in wild-type cells*. These observations led Cox and Walteld to propose a ‘biphasic’ model in which yeast cells initially activate the small amount of preformed Haclp” present to initiate the UPR, either by stabilization or another modification rendering it more transcriptionally active, and then

*Note added in proof: K. Mori and co-workers have also identified Hacl p (Em4p) as the transcription factor that activates the UPR [Mori et 01. (1996) Genes Cells 1,803-8171. They have come to similar conclusions regarding unconventional splicing of HAC7 mRNA leading to the production of active transcription factor (K. Mori, pers. commun.). Furthermore, their results suggest that retention of the unspliced mRNA in the nucleus, rather than protein degradation, may be the main mechanism regulating the expression of Hacl p.

51

sustain this response by producing the more stable Haclp’ through inducible mRNA splicing6. Genetic analyses have identified a Ser/Thr kinase (Irelp/Ernlp) whose activity is required for induction of the UPR in yeast8ng. help acts upstream of Haclp since the ZX4Cl mRNA is not spliced in yeast strains lacking the kInase6. Because its structure is similar to that of mammalian transmembrane receptor kinases, the following model for help activation has been proposed4tg: the kinase resides in the membrane of the ER or nucleus with its N-terminus facing the lumen. This domain acts as a sensor of ER stress. Mammalian receptor kinases are activated by dimerization, and Shamu and Walter have recently shown that Irelp activity is regulated similarlylO. Under nonstressed conditions, help is present as an inactive monomer. During periods of ER stress, Irelp oligomerizes, activating the kinase that nuns autophosphorylates and then induces the UPR signalling cascade. In support of this model, C-terminally truncated help molecules, which lack the kinase domain, exert a dominantnegative effectg. These mutants most probably form kinase-inactive heterodimers with wild-type Irelp. Although the mechanism of Irelp-mediated activation of the UPR is not yet clear, sequence homologies led Sidrauski et aI.’ to speculate that help itself may be the endonuclease involved in altemative splicing of the HACK mRNA. It has been suggested that the GRP78/BiP protein itself acts as a sensor of ER stress. Because GRP78/BiP binds to unfolded proteins, thereby preventing their exit from the ER, the amount of free BiP may correlate inversely with the extent of protein misfolding. In the simplest model, GRP78/BiP serves directly as a ligand for monomeric Irelp, thus preventing oligomerization of the kinase. As unfolded proteins accumulate, most GRP78/BiP will form novel complexes with malfolded proteins, thus allowing Irelp to oligomerize. Of course, many other ligands can be hypothesized and must await experimental verification. In yeast, inositol-containing phospholipids and lip ids constitute a major proportion of membrane components, and the concentration of free inositol regulates phospholipid biosynthesis”. Interestingly, yeast strains carrying mutations in IREl, HACl and RGLl are inositol auxotrophs: they require inositol in the culture medium for growth7t8.It is possible,therefore, that the UPRcoordinately regulatesboth the synthesis of ER membranes and the transcription of ERresident proteins. This would allow cells to expand the size of their ER during stress.However, it is also possiblethat Inositol starvation producesintracellular stressthat results in protein malfolding and that the UPRis required for viability under theseconditions, as it is in other circumstancesthat produce ER stress8,g. The UPR in mammalian

cells

Induction of the mammalian GRPgenesis caused by a variety of agents,including glycosylation inhibitors, reducing agents, heavy metals, amino acid analogues, glucosestarvation and perturbation of intracellular Ca2+homeostasis.It hasbeen shown that the common condition causedby all these treatments is the accumulation of malfolded proteins in the ER

52

and that this representsthe UPR-inducing stimulus3. However, the mediators of UPR activation in mammalian cells remain poorly characterized. The promoters of genesencoding mammalian GRPs contain a conserved 28-bp DNA element, the GRP core12.This sequence has been shown to complex proteins in electrophoretic mobility-shift assaysand DNA footprinting analyses. However, to date, only one constitutive factor, p70CORE, hasbeen partially purified and shown to bind to this regionin the human GRP78 promoter13. Its binding is unaffected by ER stressand its functional significance remainsto be investigated. Despite the fact that the GRPcore shares 50% overall homology to the yeast 22-bp UPREand contains a stretch of 80% identity, this sequencedoes not function asa UPRE(Ref. 5). Deletion analysisand linker scanningof the rat GRP78promoter showedthat the GRPcore element is functionally redundant14.Its deletion or mutation does not alter gene induction by malfolded proteins. Deletion of a CCAAT motif, which binds membersof the constitutive CTF/NF-1 family, reducesboth basaltranscription and stressinducibility of the rat GRP78promoter. However, this element is not sufficient for promoter activity on its own. Therefore, in mammalian cells,no singleelement seemsresponsible for ER stress-mediatedinduction of transcription; rather, this responseis mediated by multiple, functionally redundant elements14. The identification of the lrelp kinasein yeast raised the question whether the mammalian UPR is also mediatedby a kinase,perhapseven by an Irelp home logue. Cao et al. l5 investigated this hypothesis by preparing stably transfected CHO cell lines overexpressingyeast Irelp. Basaland thapsigargin-induced expression of GRP78 and GRP94was only increased twofold in these cells. Treatment with okadaic acid, a Ser/Thr phosphatase inhibitor, increased GRPinduction in responseto thapsigargin by twofold. By contrast, the tyrosine kinaseinhibitor genistein completely inhibited induction of GRP transcription by thapsigargin. Therefore, the mammalian UPRmay require both Ser/Thr and Tyr protein kinases,but these appear not to be functionally homologous to Irelp. The ER-overload

pathway

The inducible transcription factor NF-KB is an important mediator of the human immune and inflammatory response16.In most cell types, NF-KB is sequestered in an inactive, cytoplasmic complex by binding to IKB, an inhibitory subunit” (seeFig. lb). Exposure of cells to a wide variety of pathological stimuli, such asbacterial or viral infection, inflammatory cytokines or W irradiation, activates the transcription factoi?. Active NF-KB is rapidly released from the cytoplasmic complex by phosphorylationcontrolled degradation of IKB1a22(seeFig. lb). The transcription factor isthen translocatedto the nucleus, where it activates transcription of a large variety of genesincluding those of cytokines, haematopoietic growth factors and cell-adhesion molecules (for a complete list, seeRef. 16). Many ERstress-elicitingagentsthat activate the UPR activate NF-KBaswe1123,24, for example overexpression of immunoglobulin u heavy chains and treatment of trends in CELL BIOLOGY

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cells with tunicamycin, 2-deoxyglucose, brefeldin A or thapsigargln. It was tempting, therefore, to speculate that this factor may mediate the UPR in mammalian cells. However, there are a number of inducers that distinguish between UPR and NF-KB activation. Castanospermine and dithiothreitol, strong inducers of GRP expression, do not activate NF-KB. Moreover, GRPs are induced by Ca2+ ionophores25,26 and by 2-mercaptoethano127, but Ca2+ ionophores only have a co-stimulatory effect on NF-KB in T cells28, while 2-mercaptoethanol actually inhibits NF-KB activation2g. Conversely, there are inducers of NF-KB, such as tumour necrosis factor (TNF), or overexpression of the ~65 NF-KB subunit, that do not induce GRP expression but activate NF-i&-regulated genes. In addition, GRP induction depends on novel protein synthesis as it is inhibited in the presence of cycloheximide2’j30, but, since NF-KB activation is independent of protein synthesis, its activation is not blocked and is in fact enhanced by cycloheximide31. Likewise, NF-KB is activated strongly by the phosphatase inhibitor okadaic acid (OA)32. Treatment with OA does not activate GRP transcription, but can potentiate GRP induction in combination with the glycosylation inhibitor thapsigargin33. ER stresseliciting agents can be divided, therefore, into three groups: those that activate both the UPR and NF-KB, those that induce only the UPR and those that activate only NF-KB (Ref. 23). These data provide compelling evidence that ER stress-elicited NF-KB activation mediates a second and novel signal-transduction pathway between the ER and the nucleus that is pharmacologically distinct from the UPR. The stressed ER must be able, therefore, to emit two distinct signals. Because overexpression of immunoglobulin u chains and of the influenza haemagglutinin protein, both glycoproteins processed through the ER, but not of the bacterial chloramphenicol acetyl transferase (cytosolic) or firefly luclferase (peroxisomal) induces NF-KB (Refs 23 and 34), we proposed that ER overload, the congestion of this organelle with membrane proteins, triggers NF-KB activation23. By blocking protein exit from the ER, a number of ER stress-inducing agents would also cause ER overload.

However, there is a stringent requirement for ER retention. Two mutant proteins that escapeERretention and areexpressedon the cell surfaceno longer activate the transcription factor even when highly overexpressed24. This identifies the NF-r&-activating signal asthe accumulation of proteins in the ERmembrane. Ddning

the signals

emitted

by ER overload

ERoverload must releasea signalfrom the organelle that reachesNF-r&l, which residesin the cytosol. Pharmacologicalevidence suggeststhat the efflux of Ca2+ from the ERisrequiredfor ERoverload-mediatedNF-KB activation24,3g (seeFig. lb). NF-KBinduction by ERstress is prevented by preincubation of cellswith the intracellular Ca2+chelatorsTMB-8 and BAPTA-AM. Moreover, inhibition of the ER-residentCa2+-ATPase by thapsigargln or cyclopiazonic acid, which causesa rapid efflux of Ca2+from the ER, potently induces NF-KB(Ref. 24). How a changein the protein :lipid ratio of the ERmembraneincreasesCa2+permeability isunknown. Because of the incredibly steepCa2+concentration gradient over the ER membrane, a physical membrane alteration may be sufficient to change profoundly the balance between releaseand energy-dependent re-uptake of the cation. Alternatively, an accumulation of membrane proteins may impair Ca2+-ATPasefunction. Both scenarioswould explain how a number of membrane proteins that are completely unrelated in their primary structure can activate the samepathway. Ca2+is not the sole signal required for NF-KB activation by ERoverload. As observedwith many other conditions activating this factor, reactive oxygen intermediates (ROIs) alsoplay a role. Pretreatment of cells with various antioxidants abolishesNF-KBactivation by ER stress23,34,3g. However, the mechanism by which Ca2+releasetriggers the production of ROIs in responseto ER stressremains unclear. ER-nucleus

signalling

by SREBPs

Cellscan obtain cholesterol, an essentialcell membrane component, in two ways. One is the uptake of cholesterol-rich low-density lipoproteins (LDLs) by LDL receptors, which bind and internalize LDL from the plasma. Alternatively, almost all cell types can synthesize cholesterol den~vcr~~. Both cholesterol uptake and synthesisare subject to feedback repression. Defining the primary stimulus for ER overload The ER-overload hypothesis wasinvestigated using Transcription of the genesencoding the LDL receptor the adenovirus E3/19K protein asa model. Wild-type and enzymes for cholesterol biosynthesis are inhibE3/19K residesin the ERmembrane where it binds to ited by high intracellular sterol concentrations and induced upon sterol depletion4i. This requiresa lo-bp MHC classI molecules,preventing their transport to the cell surface35*36. The viral protein possesses a reten- DNA sequence,the sterol regulatory element 1 (SRE-1) tion signal sequencein its C-terminus, which causes in the promoters of sterol-regulatedgenes42. Although the protein to be retrieved continuously to the ER mutation of the SRE-1abolishes sterol responsivefrom post-ERcompartments37,38. Expressionof E3/19K ness,SRE-1cannot function efficiently on its own43,44. strongly activates NF-KB (Ref. 24). Since the sequence In the LDL receptor promoter, two adjacent binding requirements of E3/19K for MHC classI binding and sites for the ubiquitous transcription factor Spl are ERretention areknown precisely,the NF-i&-activating required for SREfunction43,45. The geneencoding SREBP-1, a basichelix-loophellx ERsignal could be investigated using point mutations leucine zipper (bHLH-LZ) protein, was cloned rethat abolish either property. It was shown that two point mutants that no cently and the protein shown to activate transcription longer bind MHC classI molecules activate NF-KBas via the SRE-1(Ref. 46). The SRE-1sequencedoesnot effectively asthe wild-type protein24.Thus, the interfit the CANNTG consensus,the so-calledE-box, deaction between E3/19K and another ER-residentpro- scribedfor other bHLH-LZ proteins. Interestingly, the gene for SREBP-1was cloned independently from an tein, MHC classI, isnot necessaryfor NF-KBactivation. trends

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expression library by virtue of the ability of the protein to bind the E-box motifq’. Spiegelmann and colleagues, who named the protein adipocyte determinationand differentiation-dependent factor 1 (ADD-l), show that the ability of SREBP-l/ADD-l to bind both the SRE and the E-box depends on a unique tyrosine residue at position 335 (Ref. 48). Mutation of this amino acid to an arginine, conserved in other bHLH-LZ proteins, abolishes binding to the SRE but retains E-box binding. Conversely, substitution of the consensus arginine by a tyrosine in another bHLH-LZ protein called USF confers the ability to bind both sequences upon this heterologous proteina. SREBP-1 and the homologous SREBP-2 (Ref. 49) are synthesized as large precursor molecules, which are found inserted into the ER membranes0 (see Fig. lc). Upon sterol depletion, SREBP precursors are cleaved by two sequential proteolytic stepsslt, releasing an -500 amino acid fragment that contains the basic DNA-binding domain as well as the leucine zipper dimerization motif. Cleaved SREBP translocates to the nucleus, where it activates transcription of target genes. In addition to the bHLH-LZ domains, SREBP/ ADD-l contains an acidic region ln its N-terminus, two long hydrophobic sequences, which represent potential transmembrane domains, and a long C-terminus preceded by Ser/Gly/Pro-rich sequencess2. By using sealed membrane vesicles, the probable orientation of SREBP-2 insertion in the ER membrane was elucidated recently 53. It appears that the protein forms a hairpin structure where both the N- and the Gtermini face the cytoplasm and a short 30-amino-acid loop, the ‘lumenal loop’, protrudes into the ER (see Fig. lc). The gene encoding a cysteine protease called SCA was cloned and the protein shown to cleave SREBP-l/ ADD-l between the leucine zipper and the first transmembrane domain in vitros4. SCA is a member of the ICE family of proteases and is the hamster homologue of the human CPP32 protein. However, it is not yet + Note added clear whether SCAKPP32 cleaves SREBP/ADDl in viva A recent study has shown very elegantly the requirein proof: cleavage of SREBP in A protein that is ment of a first sterol-dependent the ER lumen followed by a second sterol-independent required for cleaving SREBP in cleavage of SREBPin the first transmembrane domainsl. the ER membrane The nature of the proteases involved remains unknown. has recently been SRE-1 sites were first reported in the promoters of identified [Hua, X. the genes encoding the LDL receptor and HMGCoA et al. (1996) Cell 87,41M26]. This synthase. However, it has become clear recently that SREBP participates in the transcription of many more SREBP cleavagegenes. For example, the fatty acid synthase (FA5’) activating protein (ZAP) is itself a gene, which is expressed in a differentiation-dependent membrane protein, manner in adipocytes, is activated by a SREBP/ADD-1 and the binding site in its promoter4’. Together with the transmembrane observation that SREBP/ADD-1 mRNA expression insegments may be creases substantially during adipocyte differentiation, required for regulation of its these facts raise the possibility that the transcription factor exerts a dual function in regulating both sterol acti by sterols. SP AP contains no responsiveness and adipocyte differentiation-specific protease motifs, gene expression4’. and homologies Unfortunately, for most people worried about their suggest that it figure, fat cells can also synthesize lipids from glucose most likely or other carbohydrates. Genes required for this proregulates the cess, for instance the S,, gene, contain a carbohydrateSREBP-cleaving response element, a classical E-box motif, in their protease. 54

promoterss5. SREBP/ADD-1 was shown to bind and activate transcription through this element in the S,, gene48. It has been suggested that, because LDLs contain both triglycerides and cholesterol, their synthesis may be linked by the use of a single transcription factor to provide a balanced supply of both components. A similar hypothesis was made concerning a role for SREBP in maintaining a balance between amounts of fatty acid and cholesterol. It was shown recently that SREBP activates transcription from the promoter for acetyl coenzyme A carboxylase (ACC), the ratelimiting enzyme in fatty acid biosynthesisY. Since the transcription of the gene encoding HMGCoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, is also regulated by sterols, the amounts of these two cellular lipids may be regulated coordinately. Perhaps SREBP/ADD-1 also regulates membrane biosynthesis as both cholesterol and fatty acids are essential membrane components. Interestingly, the SREBP/ADD-1 binding site in the ACC promoter shows only weak homology to SRE-1. While SRE-1 consists of a contiguous sequence of 10 bp, the site in the ACC promoter consists of two non-canonical ‘half sites’, spaced 10 bp apart. Each of the half sites is only 80% homologous to the SRE-1 in the LDL receptor promoter 56. Like the LDL receptor promoter, the ACC promoter also contains a binding site for Spl adjacent to the SREBP binding site. Spl and SREBP synergistically activate the gene for the LDL receptor, an effect that may overcome the relatively low affinity of SREBP for non-consensus DNA-binding sites43. In addition to the SRE-1, the E-box and the ACC promoter site, SREBP was recently shown to bind to a fourth sequence, named SRE-3 (Ref. 57). This sequence, which is found in the promoter of the gene for farnesyl diphosphate synthase (FPP), shows only 600/6identity to either the SRE-1 or the E-box. These data raise the possibility that SREBP may be involved in the transcription of a plethora of genes mediating cholesterol, fatty acid and lipid biosynthesis, which do not contain consensus SRE-1 binding sites. For example, it has been pointed out that 13 genes encoding enzymes for lipid biosynthesis contain a conserved sequence named UAS,,, which bears a 600/6 sequence homology to both SRE-1 and the E-boxs7. In summary, the study of SREBP has revealed the central role of the ER in connecting lipid and sterol metabolism with nuclear gene expression. Conclusions

We now know three signalsthat originate from the ER and elicit a transcriptional responsein the cell nucleus: first, accumulation of unfolded (lumenal) proteins; second, accumulation of properly folded membrane proteins; and, third, changesin the lipid composition of the ERmembrane. Each of thesestimuli leads to the activation of a different signalling pathway, involving different secondmessengersand distinct transcriptional activators (Fig. la-c). Consequently, distinct sets of genes are activated, whose expressedproteins will change the phenotype of the cell in different ways. In the caseof the UPR, newly expressedproteins help the organelle to cope with improperly folded proteins in the ER.In the overload trends in CELL BIOLOGY

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response, a well-established inflammatory pathway is triggered that eventually helps the cell to mount an antiviral response. In the case of sterol depletion, a latent transcription factor that upregulates genes involved in sterol synthesis and uptake is activated by proteolysis. The characterization of these three pathways raises many new questions. For example, is HACK the only mRNA spliced by a spliceosome-independent mechanism or is tRNA ligase involved in other inducible splicing reactions as well? More importantly, are mammalian signal-transduction pathways also regulated by inducible mRNA splicing? How is the tRNA ligase regulated in response to Irelp activation in the ER membrane? Can the ER-overload response also be triggered by the accumulation of ER lumenal proteins or is a transmembrane domain required, perhaps even sufficient? How is the protease that makes the first ‘clip’ into the SREBPprecursor regulated by sterol depletion? As we come to understand more about ER-nucleus signal transduction, these pathways may become paradigms for many different cellular signalling events. References 1 LEE, S. S. et 01. (1995) Mol. Cell. Viol. 15, 3012-3022 2 VARANASI, U., CHU, R., HUANC, Q., CASTELLON, R., YELDANI, A. V. and REDDY, J. K. (1996)). Biol. Chem. 271, 2147-2155 3 KOZUTSUMI, Y., SEGAL, M., NORMINGTON, K., GETHING, M-J. and SAMBROOK, J. (1988) Nature 332,462-464 4 SHAMU, C. E., COX, 1. S. and WALTER, P. (1994) Trends Cell Biol. 4, 57-60 5 MORI, K., SANT, A., KOHNO, K., NORMINGTON, K., CETHING, M-J. and SAMBROOK, J. F. (1992) EMBO /. 11, 2583-2593 6 COX, J. S. and WALTER, P. (1996) Cell 87, 391-404 7 SIDRAUSKI, C., COX, J. 5. and WALTER, P. (1996) Ce//87,405-413 8 COX, J. S., SHAMU, C. E. and WALTER, P. (1993) Cell 73, 1197-l 206 9 MORI, K., MA, W., CETHING, M-J. and SAMBROOK, J. (1993) Cell 74, 743-756 10 SHAMU, C. E. and WALTER, P. (1996) FMBO 1.15, 3028-3039 11 WHITE, M. J., LOPES, J. M. and HENRY, 5. A. (1991) Adv. Microb. Physiol. 32, 1-51 12 CHANC, S. C., ERWIN, A. E. and LEE, A. S. (1989) Mol. Cell. Biol. 9,2153-2162 13 LI, W. W., SISTONEN, L., MORIMOTO, R. I. and LEE, A. 5. (1994) Mol. Cell. Biol. 14, 5533-5546 14 WOODEN, S. K., LI, L-J., NAVARRO, D., QADRI, I., PEREIRA, L. and LEE, A. 5. (1991) Mol. Cell. Biol. 11, 5612-5623 15 CAO, X., ZHOU, Y. and LEE, A. S. (1995) 1. Biol. Chem. 270, 494-502 16 BAEUERLE, P. A. and HENKEL, T. (1994) Annu. Rev. Immunol. 12,141-179 17 BAEUERLE, P. A. and BALTIMORE, D. (1988) Ce1153,21 l-207 18 BEG, A. A., FINCO, T. 5, NANTERMET, P. V. and BALDWIN, A. S., Jr (1993) Mol. Cell. Biol. 13, 3301-3310 19 HENKEL, T., MACHLEIDT, T., ALKAIAY I., KRljNKE, M., BEN-NERIAH, Y. and BAEUERLE, P. A. (1993) Nature 365,182-l 84 20 SUN, S-C., GANCHI, P. A., BhAUD, C., BALLARD, D. W. and GREENE, W. C. (1994) Proc. Not/. Acad. Sci. U. 5. A. 87, 1346-I 350 21 TRAENCKNER, E. B-M., PAHL, H. L., HENKEL, T., SCHMIDT, K. N., WILK, S. and BAEUERLE, P. A. (1995) trends in CELL BIOLOGY

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