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Membrane-bound transcription factors: regulated release by RIP or RUP
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Membrane-bound transcription factors: regulated release by RIP or RUP Thorsten Hoppe*†, Michael Rape* and Stefan Jentsch* Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membranebound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane. Addresses *Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany † Present address: Gene Centre, University of Munich, Feodor-LynenStr. 25, 81377 Munich, Germany Correspondence: Stefan Jentsch; e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:344–348 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ER endoplasmic reticulum ERAD ER-associated protein degradation OLE pathway that controls OLE1 desaturase levels RIP regulated intramembrane proteolysis RUP regulated ubiquitin/proteasome-dependent processing S1P Site-1 protease SCAP SREBP cleavage-activating protein SRE serum response element SREBP SRE-binding protein UBC ubiquitin-conjugating enzyme UFA unsaturated fatty acid UPR unfolded protein response
Introduction The magical awakening of a dormant benefactor is a perpetual theme of many ancient traditions and folk tales. But within cells similar episodes are a reality. Quite a few regulatory proteins, including transcription factors, are normally kept in a dormant state and are activated by internal or environmental cues. Examples of these include the STAT transcription factors, which are stored in the cytosol and are ‘kissed’ to life by JAK-mediated phosphorylation, and NF-κB p50, which is prevented from entering the nucleus until its cytosolic captor is destroyed (reviewed in [1,2]). Indeed, as illustrated in these examples, many of the activation steps control early steps of nuclear transport pathways of transcription factors. Signal-induced activation of dormant, cytosolic pools of transcription factors seems to be an exquisite process that warrants a rapid transcriptional response. Recently, a novel strategy for the mobilisation of dormant transcription factors was discovered that requires proteolytic
cleavage. Transcription factors have been identified that are initially made as inactive, membrane-bound precursors. Once triggered by intracellular cues, these proteins are cleaved and the vitalised transcription factors can migrate into the nucleus to drive transcription. Two fundamentally different cleavage mechanisms have been identified that have been coined ‘RIP’ (regulated intramembrane proteolysis) and ‘RUP’ (regulated ubiquitin/proteasome-dependent processing). These pathways involve membrane-bound, site-specific proteases or the proteasome, the versatile proteolysis machinery of the cell, respectively. In this review we will discuss these two fascinating pathways and argue that these systems are perfectly suited for their biological assignments.
Regulated intramembrane proteolysis The first example of a membrane-bound transcription factor was SREBP (SRE-binding protein), which regulates sterols in vertebrate cells [3]. SREBP binds to sterol response elements (SREs) within the enhancers of sterolregulated genes and induces their transcription [4]. High levels of cholesterol lead to reduced transcription of genes encoding enzymes involved in the biosynthesis of cholesterol, unsaturated fatty acids and triglycerides, as well as genes that control lipid uptake [5]. Cholesterol depletion, on the other hand, has the opposite effect. SREBP is initially synthesized as a 125 kDa membranebound precursor with two hydrophobic transmembrane segments. Three isoforms of SREBP have been identified so far [6,7], and they share a similar tripartite structure: an amino-terminal region, which possesses a transcription factor domain of the basic helix-loop-helix-leucine zipper (bHLHZip) family; a central domain, which contains the two transmembrane spans; and a carboxy-terminal regulatory domain [8,9]. SREBP inserts into the ER and/or the nuclear envelope in a hairpin-like fashion, thereby exposing both the amino- and carboxy-terminal domains to the cytosol [10,11]. SREBP activation is accomplished by two sequential proteolytic cleavages of the 125 kDa precursor form of SREBP, thereby liberating the 65 kDa amino-terminal transcription factor domain, which can subsequently migrate into the nucleus. The two cleavages are mediated by distinct sitespecific proteases, but, interestingly, only the first cleavage is directly regulated by cholesterol. The first cut occurs at a site within the hydrophilic loop of the SREBP precursor within the lumen. It is performed by the so-called Site-1 protease (S1P), a subtilisin-related serine protease, which is membrane-bound and oriented with its active site pointing towards the lumen [12]. This cleavage yields an amino-terminal and a carboxy-terminal SREBP intermediate and allows the second protease, Site-2 protease (S2P), to cleave
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Figure 1 (a) The 125 kDa inactive SREBP-precursor inserts into the ER and/or the nuclear envelope in a hairpin-like fashion, with the amino- and carboxy terminal domains facing the cytosol. At low levels of cholesterol in the ER-membrane, SREBP binds to SCAP, which carries SREBP piggyback to a post-ER/Golgi-compartment (1). There it meets active Site-1 protease (S1P) and SREBP receives its first cleavage (2). It is currently not known whether the second cleavage of SREBP by Site-2 protease (S2P) occurs in the same compartment or whether SREBP has to move back to the ER to receive its second cleavage (3). The second cleavage occurs within the transmembrane span of SREBP, thereby releasing the 65 kDa active transcription factor for nuclear transport and transcription of target genes (4). (b) The 120 kDa inactive SPT23 precursor inserts into the ER-membrane with the bulk of the protein facing the cytosol. SPT23 interacts with the RSP5 ubiquitin ligase (E3-enzyme) and gets ubiquitinated (1). The ubiquitinated SPT23precursor is then processed by the 26S proteasome (2). Whereas the carboxy-terminal portion of SPT23, including its transmembrane span, is completely degraded, the amino-terminal transcription factor domain (p90) is spared from degradation and is released into the cytosol (3). The active 90 kDa transcription factor then migrates into the nucleus and activates the transcription of the OLE1-gene (4).
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off the transcription factor domain from the amino-terminal S1P cleavage product. Intriguingly, S2P is a membraneembedded zinc metalloprotease that has its catalytic site located within the plane of the membrane and cleaves SREBP within its transmembrane span [13,14]. Processing of SREBP is blocked by sterols, which selectively inhibit the first cleavage by S1P. S2P is only indirectly regulated by sterols because the protease cannot act before the initial cleavage by S1P has occurred. Sterolmediated regulation of SREBP processing depends on the SREBP cleavage-activating protein (SCAP) [15]. SCAP is an integral membrane protein with two distinct domains [16]: the amino-terminal domain, which contains eight membrane-spanning helices that anchor SCAP to the ER membrane, and the carboxy-terminal domain, which is exposed to the cytosol and contains five WD40 repeats. Interaction between the WD40 repeat domain of SCAP and the carboxy-terminal regulatory domain of SREBP is required for SREBP cleavage by S1P [12,17]. SCAP also contains a sterol-sensing domain, which might be responsible for the SCAP-mediated sterol regulation of SREBP cleavage by S1P [4]. In contrast to the uncleaved precursor form of SREBP, which is bound to the ER membrane [3,11], the active form of S1P is localised in a post-ER/Golgi compartment
Current Opinion in Cell Biology
[18,19]. Moreover, SCAP moves from the ER to the postER/Golgi compartment, and this movement may be regulated by sterols [20]. These findings have led to the model that SCAP carries SREBP ‘piggyback’ from the ER to the Golgi and that this transport is blocked by cholesterol [18,21]. SREBP then meets active S1P in the Golgi, and the SREBP precursor receives its first cleavage. It is currently not known whether the second cleavage occurs within the same post-ER compartment or whether cleaved SREBP has to travel back to the ER to meet the second protease, S2P (see Figure 1a). Because the cleavage by S2P occurs within the transmembrane span of SREBP, this mechanism has been dubbed regulated intramembrane proteolysis or RIP. Several other cellular pathways are known or postulated to be controlled by RIP [22]. Processing of the IRE1 protein kinase and the ATF6 transcription factor, two transmembrane proteins implicated in the unfolded protein response (UPR), was suggested to occur via RIP ([23,24•]; see also the review by Patil and Walter pp 349-355 of this issue). Both proteins are cleaved, and thereby activated, upon ER-stress conditions. Interestingly, processing of ATF6 is mediated by the same proteases that cleave SREBP, but this reaction is neither regulated by cholesterol nor does it require SCAP [25••]. Whether ATF6, which bears a single transmembrane span, also has to move to a post-ER/Golgi
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compartment for cleavage, and if so, if a SCAP analog is required, is presently not known. RIP may also act on Notch, a transmembrane signalling protein controlling development [26]. In this case, binding of a Notch ligand induces RIP, thereby releasing an active cytosolic domain of Notch. The amyloid precursor protein (APP) is also cleaved post-translationally within the ER and Golgi membranes [27]. The resulting product, the toxic amyloid β peptide, is involved in the development of Alzheimer’s disease. Cleavage of APP and Notch does not seem to be mediated by S1P or S2P but instead requires presenilin, a membrane protein that might function as a protease (γ-secretase) [28•]. Whether mammalian IRE1 cleavage is also mediated by presenilin is currently a matter of debate [23, 29]. RIP is not a phenomenon restricted to animal cells — it has also been observed in bacteria. For example, sporulation of Bacillus subtilis requires the protease SpoIVFB [30], a bacterial homolog of S2P [22]. This enzyme cleaves the membrane-bound factor pro-σK, thereby liberating σK into the cytosol of the mother cell where it activates late sporulation genes of the mother cell [31].
Regulated ubiquitin/proteasome-dependent processing Recently, processing of membrane-bound transcription factors was discovered in the yeast Saccharomyces cerevisiae; however, the cleavage mechanism turned out to be a surprise [32••]. Two homologous transcription factors, SPT23 and MGA2, are crucial components of a regulatory pathway, termed the OLE pathway, that controls membrane fluidity in yeast. SPT23 and MGA2 are required for the expression of OLE1, which encodes the enzyme ∆9 fatty acid desaturase [33]. This ER-bound enzyme catalyses the desaturation of C16 and C18 fatty acids to form the monounsaturated fatty acids (UFAs) palmitoleic (16:1) and oleic acid (18:1). The presence of UFAs is essential for cell growth and crucially important for normal membrane fluidity. SPT23 and MGA2 are structurally and functionally related to each other and are distant homologs of the NF-κB/Rel transcription factors of higher eukaryotes. In contrast to NF-κB/Rel, both yeast transcription factors possess a single carboxy-terminal transmembrane domain. The proteins are anchored to the ER in such a way that the bulk of the proteins faces the cytosol ([32••]; M Rape, T Hoppe, S Jentsch, unpublished data). The full-length, membrane-bound forms (p120) of SPT23 and MGA2 are inactive, but a proteolytic cleavage event liberates the active transcription factors (p90), which can subsequently migrate into the nucleus to drive transcription (see Figure 1b). Intriguingly, SPT23 (and MGA2) processing was found to be mediated by the ubiquitin/proteasome pathway [32••]. Membrane-bound SPT23 p120 interacts with a specific WW-(protein interaction) domain of the RSP5 ubiquitin ligase (an E3 enzyme) [34].
Ubiquitination of the precursor then triggers SPT23 processing, which is mediated by the 26S proteasome. The carboxy-terminal portion of SPT23, including the transmembrane span, appears to be completely degraded by the proteasome in a process similar to ER-associated degradation (ERAD) of membrane proteins [35]. In contrast, the amino-terminal 90 kDa transcription factor domain (p90) is spared from degradation and is released into the cytosol. Mobilisation of p90 for nuclear import additionally requires the chaperone-like enzyme CDC48, an AAA-type ATPase, and two other proteins, UFD1 and NPL4 ([32••]; M Rape, T Hoppe, S Jentsch, unpublished data). The OLE pathway is tightly regulated at several levels [36,37]. Remarkably, SPT23 processing is regulated by UFAs: addition of UFAs (16:1, 18:2 and 18:3) into the growth medium completely abolishes SPT23 processing [32••]. This finding indicates that the membrane-bound precursor of SPT23 may act as a sensor for membrane fluidity or thickness. Regulation of SPT23 processing presumably acts at an early step in the OLE pathway, probably by controlling the SPT23–RSP5 interaction. Although SPT23 and MGA2 do not seem to have orthologs in mammalian cells, the mechanism of processing, termed ‘ubiquitin/proteasome-dependent processing’ or RUP, is conserved. One example is the processing of p105, the cytosolic precursor of the p50 subunit of the NF-κB transcription factor. NF-κB plays key roles in basic processes such as regulation of the immune and inflammatory responses, development, malignant transformation and apoptosis [2]. Processing of ubiquitinated p105 leads to the rapid degradation of its carboxy-terminal portion by the proteasome, whereas the amino-terminal p50 part remains stable [38,39]. The precursors of SPT23/MGA2 (p120) and NF-κB (p105) exhibit a strikingly cognate organisation and possess similar protein domains, including immunoglobulinlike putative DNA-binding domains and several ankyrin repeats [32••]. Given this similarity it seems likely that the mechanism of proteasome-dependent processing works for both proteins by a conserved mechanism. Because SPT23 p120 is anchored to membranes via its carboxy-terminal tail we proposed that a polypeptide loop of SPT23 enters the barrel-shaped 26S proteasome to contact the active sites within the protease [32••]. Indeed, the dimensions of the proteasome would allow the accommodation of a polypeptide loop [40]. We further hypothesize that the proteasome will processively degrade the carboxy-terminal portions of p120 and p105 but that the amino-terminal active domains of both proteins are spared from degradation because they are tightly bound to other proteins, for example, another precursor molecule. Disruption of this interaction would then mobilise the transcription factors for nuclear import. Another likely example for RUP is the processing of the transcriptional regulatory protein Cubitus interruptus (Ci), a component of the Hedgehog (Hh) signaling pathway [41]. Hh is a secreted morphogen that directs a
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variety of developmental events in vertebrates and flies. Hh signalling regulates the transcription of several tissuespecific target genes, which is largely controlled by Ci. In the absence of Hh signalling, full-length Ci (Ci-155) gets proteolytically processed and the 75 kDa amino-terminal portion (Ci-75) remains intact and acts as a transcriptional repressor [42]. In contrast, in the presence of the Hh protein, Ci processing is suppressed and the intact Ci protein (Ci-155) acts as a transcriptional activator of Hh target genes. Ci-processing seems to involve ubiquitination via the Slimb F-box protein and proteasomes [43•,44•], suggesting that the processing reaction is indeed mediated by RUP.
Conclusions Rupture by RIP and RUP must now be added to the list of cellular tricks that regulate nuclear import and gene expression. Both mechanisms are well suited for a rapid and reversible activation of a gene expression program. Why nature has evolved two very different strategies for similar reactions is not clear in the moment, but future studies are expected to reveal the specific potencies of the two pathways. The examples discussed in this review also make it clear that RIP and RUP are not restricted to the activation of transcription factors — not even the regulation of nuclear import. In fact, cleaving off biologically active protein domains from precursors appears to be a very versatile biological concept. Both types of mechanisms, site-specific cleavage of membrane spans and the partial degradation of a protein by the proteasome, are fascinating strategies. Yet perhaps even more exciting is the question how regulatory circuits like the SREBP and the OLE pathway are controlled. Regulatory circuits typically require specific tactics to turn them on and to shut them down. It turns out that SREBP and SPT23 are extremely well suited for their biological tasks. In both cases the ER-bound precursors of these transcription factors receive their signals from their immediate environment, the ER-membrane. Cleavage of SREBP is induced by low cholesterol levels in the membrane, whereas SPT23 cleavage may be induced by membrane fluiditycontrolled interaction with the RSP5 ubiquitin ligase. Similarly, ER-bound ATF6, which controls the ER stress response (UPR), is presumably activated by unfolded proteins within the lumen of the ER. The idea that further cues, for example, signals transduced from sensors for temperature and cell growth, trigger SREBP or SPT23 processing is speculative. But how are the regulatory circuits turned off? First, cleavage of SREBP and SPT23 are directly repressed by high levels of cholesterol and UFAs, respectively. Second, the mature forms of SREBP, SPT23 and ATF6 transcription factors are very short lived. Indeed, both proteins appear to be degraded in the nucleus by proteasomes ([3,25•,32••]; M Rape, T Hoppe, S Jentsch, unpublished data). Although several details concerning these two regulation circuits have to await future studies, the two tales of SREBP and SPT23 make it amply clear that membrane-bound transcription factors are
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fabulously qualified for transmitting signals from the vicinity of a cellular membrane to the nucleus. Hence, it will be only a matter of time before we find out what other examples nature has in store.
Acknowledgements We thank Alexander Buchberger for discussions and comments.
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Sakai J, Nohturfft A, Cheng D, Ho YK, Brown MS, Goldstein JL: Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein. J Biol Chem 1997, 272:20213-20221.
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18. DeBose-Boyd RA, Brown MS, Li WP, Nohturfft A, Goldstein JL, Espenshade PJ: Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi. Cell 1999, 99:703-712.
32. Hoppe T, Matuschewski K, Rape M, Schlenker S, Ulrich HD, •• Jentsch S: Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing. Cell 2000, 102:577-586. This study reports that yeast homologs of NF-κB are initially made as membrane-bound precursors. The proteins were found to be processed by regulated ubiquitin/proteasome-dependent processing (RUP rather than by RIP).
19. Espenshade PJ, Cheng D, Goldstein JL, Brown MS: Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins. J Biol Chem 1999, 274:22795-22804.
33. Zhang S, Skalsky Y, Garfinkel DJ: MGA2 or SPT23 is required for transcription of the ∆9 fatty acid desaturase gene, OLE1, and nuclear membrane integrity in Saccharomyces cerevisiae. Genetics 1999, 151:473-483.
20. Nohturfft A, DeBose-Boyd RA, Scheek S, Goldstein JL, Brown MS: Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi. Proc Natl Acad Sci USA 1999, 96:11235-11240.
34. Hochstrasser M: Ubiquitin-dependent protein degradation. Annu Rev Genet 1996, 30:405-439.
21. Nohturfft A, Yabe D, Goldstein JL, Brown MS, Espenshade PJ: Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes. Cell 2000, 102:315-323. 22. Brown MS, Ye J, Rawson RB, Goldstein JL: Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 2000, 100:391-398. 23. Niwa M, Sidrauski C, Kaufman RJ, Walter P: A role for presenilin-1 in nuclear accumulation of Ire1 fragments and induction of the mammalian unfolded protein response. Cell 1999, 99:691-702. 24. Haze K, Yoshida H, Yanagi H, Yura T, Mori K: Mammalian • transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 1999, 10:3787-3799. This work nicely extends the list of membrane-bound transcription factors regulated by RIP. 25. Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R, Brown MS, •• Goldstein JL: ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell 2000, 6:1355-1364. ATF6 is shown here to be processed by S1P and S2P but in a sterol and SCAP-independent fashion. 26. Annaert W, De Strooper B: Presenilins: molecular switches between proteolysis and signal transduction. Trends Neurosci 1999, 22:439-443. 27.
Selkoe DJ: The cell biology of β-amyloid precursor protein and presenilin in Alzheimer’s disease. Trends Cell Biol 1998, 8:447-453.
28. Wolfe MS, Xia W, Ostaszewski BL, Diehl TS, Kimberly WT, Selkoe DJ: • Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and γ-secretase activity. Nature 1999, 398:513-517. Strong evidence is given here for a direct function of presenilin as a protease. 29. Mori K: Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell 2000, 101:451-454. 30. Rudner DZ, Fawcett P, Losick R: A family of membrane-embedded metalloproteases involved in regulated proteolysis of membraneassociated transcription factors. Proc Natl Acad Sci USA 1999, 96:14765-14770.
35. Mayer TU, Braun T, Jentsch S: Role of the proteasome in membrane extraction of a short-lived ER-transmembrane protein. EMBO J 1998, 17:3251-3257. 36. Choi JY, Stukey J, Hwang SY, Martin CE: Regulatory elements that control transcription activation and unsaturated fatty acidmediated repression of the Saccharomyces cerevisiae OLE1 gene. J Biol Chem 1996, 271:3581-3589. 37.
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κB by 38. Fan CM, Maniatis T: Generation of p50 subunit of NF-κ processing of p105 through an ATP-dependent pathway. Nature 1991, 354:395-398. 39. Palombella VJ, Rando OJ, Goldberg AL, Maniatis T: The ubiquitinκB 1 proteasome pathway is required for processing the NF-κ κB. Cell 1994, precursor protein and the activation of NF-κ 78:773-785. 40. Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD, Huber R: Structure of 20S proteasome from yeast at 2.4 Å resolution. Nature 1997, 386:463-471. 41. Aza-Blanc P, Kornberg TB: Ci: a complex transducer of the hedgehog signal. Trends Genet 1999, 15:458-462. 42. Aza-Blanc P, Ramirez-Weber FA, Laget MP, Schwartz C, Kornberg TB: Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 1997, 89:1043-1053. 43. Chen CH, von Kessler DP, Park W, Wang B, Ma Y, Beachy PA: • Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell 1999, 98:305-316. This paper (along with [42]) provides strong evidence for the proteasome being involved in the processing of Ci. 44. Jiang J, Struhl G: Regulation of the Hedgehog and Wingless • signalling pathways by the F-box/WD40-repeat protein Slimb. Nature 1998, 391:493-496. As F-box proteins are subunits of E3-type ubiquitin ligases, this work suggests that Ci processing is mediated by ubiquitin/proteasome-dependent processing.
Membrane-bound transcription factors: regulated release by RIP or RUP Thorsten Hoppe*†, Michael Rape* and Stefan Jentsch* Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membranebound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane. Addresses *Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany † Present address: Gene Centre, University of Munich, Feodor-LynenStr. 25, 81377 Munich, Germany Correspondence: Stefan Jentsch; e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:344–348 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ER endoplasmic reticulum ERAD ER-associated protein degradation OLE pathway that controls OLE1 desaturase levels RIP regulated intramembrane proteolysis RUP regulated ubiquitin/proteasome-dependent processing S1P Site-1 protease SCAP SREBP cleavage-activating protein SRE serum response element SREBP SRE-binding protein UBC ubiquitin-conjugating enzyme UFA unsaturated fatty acid UPR unfolded protein response
Introduction The magical awakening of a dormant benefactor is a perpetual theme of many ancient traditions and folk tales. But within cells similar episodes are a reality. Quite a few regulatory proteins, including transcription factors, are normally kept in a dormant state and are activated by internal or environmental cues. Examples of these include the STAT transcription factors, which are stored in the cytosol and are ‘kissed’ to life by JAK-mediated phosphorylation, and NF-κB p50, which is prevented from entering the nucleus until its cytosolic captor is destroyed (reviewed in [1,2]). Indeed, as illustrated in these examples, many of the activation steps control early steps of nuclear transport pathways of transcription factors. Signal-induced activation of dormant, cytosolic pools of transcription factors seems to be an exquisite process that warrants a rapid transcriptional response. Recently, a novel strategy for the mobilisation of dormant transcription factors was discovered that requires proteolytic
cleavage. Transcription factors have been identified that are initially made as inactive, membrane-bound precursors. Once triggered by intracellular cues, these proteins are cleaved and the vitalised transcription factors can migrate into the nucleus to drive transcription. Two fundamentally different cleavage mechanisms have been identified that have been coined ‘RIP’ (regulated intramembrane proteolysis) and ‘RUP’ (regulated ubiquitin/proteasome-dependent processing). These pathways involve membrane-bound, site-specific proteases or the proteasome, the versatile proteolysis machinery of the cell, respectively. In this review we will discuss these two fascinating pathways and argue that these systems are perfectly suited for their biological assignments.
Regulated intramembrane proteolysis The first example of a membrane-bound transcription factor was SREBP (SRE-binding protein), which regulates sterols in vertebrate cells [3]. SREBP binds to sterol response elements (SREs) within the enhancers of sterolregulated genes and induces their transcription [4]. High levels of cholesterol lead to reduced transcription of genes encoding enzymes involved in the biosynthesis of cholesterol, unsaturated fatty acids and triglycerides, as well as genes that control lipid uptake [5]. Cholesterol depletion, on the other hand, has the opposite effect. SREBP is initially synthesized as a 125 kDa membranebound precursor with two hydrophobic transmembrane segments. Three isoforms of SREBP have been identified so far [6,7], and they share a similar tripartite structure: an amino-terminal region, which possesses a transcription factor domain of the basic helix-loop-helix-leucine zipper (bHLHZip) family; a central domain, which contains the two transmembrane spans; and a carboxy-terminal regulatory domain [8,9]. SREBP inserts into the ER and/or the nuclear envelope in a hairpin-like fashion, thereby exposing both the amino- and carboxy-terminal domains to the cytosol [10,11]. SREBP activation is accomplished by two sequential proteolytic cleavages of the 125 kDa precursor form of SREBP, thereby liberating the 65 kDa amino-terminal transcription factor domain, which can subsequently migrate into the nucleus. The two cleavages are mediated by distinct sitespecific proteases, but, interestingly, only the first cleavage is directly regulated by cholesterol. The first cut occurs at a site within the hydrophilic loop of the SREBP precursor within the lumen. It is performed by the so-called Site-1 protease (S1P), a subtilisin-related serine protease, which is membrane-bound and oriented with its active site pointing towards the lumen [12]. This cleavage yields an amino-terminal and a carboxy-terminal SREBP intermediate and allows the second protease, Site-2 protease (S2P), to cleave
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Figure 1 (a) The 125 kDa inactive SREBP-precursor inserts into the ER and/or the nuclear envelope in a hairpin-like fashion, with the amino- and carboxy terminal domains facing the cytosol. At low levels of cholesterol in the ER-membrane, SREBP binds to SCAP, which carries SREBP piggyback to a post-ER/Golgi-compartment (1). There it meets active Site-1 protease (S1P) and SREBP receives its first cleavage (2). It is currently not known whether the second cleavage of SREBP by Site-2 protease (S2P) occurs in the same compartment or whether SREBP has to move back to the ER to receive its second cleavage (3). The second cleavage occurs within the transmembrane span of SREBP, thereby releasing the 65 kDa active transcription factor for nuclear transport and transcription of target genes (4). (b) The 120 kDa inactive SPT23 precursor inserts into the ER-membrane with the bulk of the protein facing the cytosol. SPT23 interacts with the RSP5 ubiquitin ligase (E3-enzyme) and gets ubiquitinated (1). The ubiquitinated SPT23precursor is then processed by the 26S proteasome (2). Whereas the carboxy-terminal portion of SPT23, including its transmembrane span, is completely degraded, the amino-terminal transcription factor domain (p90) is spared from degradation and is released into the cytosol (3). The active 90 kDa transcription factor then migrates into the nucleus and activates the transcription of the OLE1-gene (4).
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off the transcription factor domain from the amino-terminal S1P cleavage product. Intriguingly, S2P is a membraneembedded zinc metalloprotease that has its catalytic site located within the plane of the membrane and cleaves SREBP within its transmembrane span [13,14]. Processing of SREBP is blocked by sterols, which selectively inhibit the first cleavage by S1P. S2P is only indirectly regulated by sterols because the protease cannot act before the initial cleavage by S1P has occurred. Sterolmediated regulation of SREBP processing depends on the SREBP cleavage-activating protein (SCAP) [15]. SCAP is an integral membrane protein with two distinct domains [16]: the amino-terminal domain, which contains eight membrane-spanning helices that anchor SCAP to the ER membrane, and the carboxy-terminal domain, which is exposed to the cytosol and contains five WD40 repeats. Interaction between the WD40 repeat domain of SCAP and the carboxy-terminal regulatory domain of SREBP is required for SREBP cleavage by S1P [12,17]. SCAP also contains a sterol-sensing domain, which might be responsible for the SCAP-mediated sterol regulation of SREBP cleavage by S1P [4]. In contrast to the uncleaved precursor form of SREBP, which is bound to the ER membrane [3,11], the active form of S1P is localised in a post-ER/Golgi compartment
Current Opinion in Cell Biology
[18,19]. Moreover, SCAP moves from the ER to the postER/Golgi compartment, and this movement may be regulated by sterols [20]. These findings have led to the model that SCAP carries SREBP ‘piggyback’ from the ER to the Golgi and that this transport is blocked by cholesterol [18,21]. SREBP then meets active S1P in the Golgi, and the SREBP precursor receives its first cleavage. It is currently not known whether the second cleavage occurs within the same post-ER compartment or whether cleaved SREBP has to travel back to the ER to meet the second protease, S2P (see Figure 1a). Because the cleavage by S2P occurs within the transmembrane span of SREBP, this mechanism has been dubbed regulated intramembrane proteolysis or RIP. Several other cellular pathways are known or postulated to be controlled by RIP [22]. Processing of the IRE1 protein kinase and the ATF6 transcription factor, two transmembrane proteins implicated in the unfolded protein response (UPR), was suggested to occur via RIP ([23,24•]; see also the review by Patil and Walter pp 349-355 of this issue). Both proteins are cleaved, and thereby activated, upon ER-stress conditions. Interestingly, processing of ATF6 is mediated by the same proteases that cleave SREBP, but this reaction is neither regulated by cholesterol nor does it require SCAP [25••]. Whether ATF6, which bears a single transmembrane span, also has to move to a post-ER/Golgi
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compartment for cleavage, and if so, if a SCAP analog is required, is presently not known. RIP may also act on Notch, a transmembrane signalling protein controlling development [26]. In this case, binding of a Notch ligand induces RIP, thereby releasing an active cytosolic domain of Notch. The amyloid precursor protein (APP) is also cleaved post-translationally within the ER and Golgi membranes [27]. The resulting product, the toxic amyloid β peptide, is involved in the development of Alzheimer’s disease. Cleavage of APP and Notch does not seem to be mediated by S1P or S2P but instead requires presenilin, a membrane protein that might function as a protease (γ-secretase) [28•]. Whether mammalian IRE1 cleavage is also mediated by presenilin is currently a matter of debate [23, 29]. RIP is not a phenomenon restricted to animal cells — it has also been observed in bacteria. For example, sporulation of Bacillus subtilis requires the protease SpoIVFB [30], a bacterial homolog of S2P [22]. This enzyme cleaves the membrane-bound factor pro-σK, thereby liberating σK into the cytosol of the mother cell where it activates late sporulation genes of the mother cell [31].
Regulated ubiquitin/proteasome-dependent processing Recently, processing of membrane-bound transcription factors was discovered in the yeast Saccharomyces cerevisiae; however, the cleavage mechanism turned out to be a surprise [32••]. Two homologous transcription factors, SPT23 and MGA2, are crucial components of a regulatory pathway, termed the OLE pathway, that controls membrane fluidity in yeast. SPT23 and MGA2 are required for the expression of OLE1, which encodes the enzyme ∆9 fatty acid desaturase [33]. This ER-bound enzyme catalyses the desaturation of C16 and C18 fatty acids to form the monounsaturated fatty acids (UFAs) palmitoleic (16:1) and oleic acid (18:1). The presence of UFAs is essential for cell growth and crucially important for normal membrane fluidity. SPT23 and MGA2 are structurally and functionally related to each other and are distant homologs of the NF-κB/Rel transcription factors of higher eukaryotes. In contrast to NF-κB/Rel, both yeast transcription factors possess a single carboxy-terminal transmembrane domain. The proteins are anchored to the ER in such a way that the bulk of the proteins faces the cytosol ([32••]; M Rape, T Hoppe, S Jentsch, unpublished data). The full-length, membrane-bound forms (p120) of SPT23 and MGA2 are inactive, but a proteolytic cleavage event liberates the active transcription factors (p90), which can subsequently migrate into the nucleus to drive transcription (see Figure 1b). Intriguingly, SPT23 (and MGA2) processing was found to be mediated by the ubiquitin/proteasome pathway [32••]. Membrane-bound SPT23 p120 interacts with a specific WW-(protein interaction) domain of the RSP5 ubiquitin ligase (an E3 enzyme) [34].
Ubiquitination of the precursor then triggers SPT23 processing, which is mediated by the 26S proteasome. The carboxy-terminal portion of SPT23, including the transmembrane span, appears to be completely degraded by the proteasome in a process similar to ER-associated degradation (ERAD) of membrane proteins [35]. In contrast, the amino-terminal 90 kDa transcription factor domain (p90) is spared from degradation and is released into the cytosol. Mobilisation of p90 for nuclear import additionally requires the chaperone-like enzyme CDC48, an AAA-type ATPase, and two other proteins, UFD1 and NPL4 ([32••]; M Rape, T Hoppe, S Jentsch, unpublished data). The OLE pathway is tightly regulated at several levels [36,37]. Remarkably, SPT23 processing is regulated by UFAs: addition of UFAs (16:1, 18:2 and 18:3) into the growth medium completely abolishes SPT23 processing [32••]. This finding indicates that the membrane-bound precursor of SPT23 may act as a sensor for membrane fluidity or thickness. Regulation of SPT23 processing presumably acts at an early step in the OLE pathway, probably by controlling the SPT23–RSP5 interaction. Although SPT23 and MGA2 do not seem to have orthologs in mammalian cells, the mechanism of processing, termed ‘ubiquitin/proteasome-dependent processing’ or RUP, is conserved. One example is the processing of p105, the cytosolic precursor of the p50 subunit of the NF-κB transcription factor. NF-κB plays key roles in basic processes such as regulation of the immune and inflammatory responses, development, malignant transformation and apoptosis [2]. Processing of ubiquitinated p105 leads to the rapid degradation of its carboxy-terminal portion by the proteasome, whereas the amino-terminal p50 part remains stable [38,39]. The precursors of SPT23/MGA2 (p120) and NF-κB (p105) exhibit a strikingly cognate organisation and possess similar protein domains, including immunoglobulinlike putative DNA-binding domains and several ankyrin repeats [32••]. Given this similarity it seems likely that the mechanism of proteasome-dependent processing works for both proteins by a conserved mechanism. Because SPT23 p120 is anchored to membranes via its carboxy-terminal tail we proposed that a polypeptide loop of SPT23 enters the barrel-shaped 26S proteasome to contact the active sites within the protease [32••]. Indeed, the dimensions of the proteasome would allow the accommodation of a polypeptide loop [40]. We further hypothesize that the proteasome will processively degrade the carboxy-terminal portions of p120 and p105 but that the amino-terminal active domains of both proteins are spared from degradation because they are tightly bound to other proteins, for example, another precursor molecule. Disruption of this interaction would then mobilise the transcription factors for nuclear import. Another likely example for RUP is the processing of the transcriptional regulatory protein Cubitus interruptus (Ci), a component of the Hedgehog (Hh) signaling pathway [41]. Hh is a secreted morphogen that directs a
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variety of developmental events in vertebrates and flies. Hh signalling regulates the transcription of several tissuespecific target genes, which is largely controlled by Ci. In the absence of Hh signalling, full-length Ci (Ci-155) gets proteolytically processed and the 75 kDa amino-terminal portion (Ci-75) remains intact and acts as a transcriptional repressor [42]. In contrast, in the presence of the Hh protein, Ci processing is suppressed and the intact Ci protein (Ci-155) acts as a transcriptional activator of Hh target genes. Ci-processing seems to involve ubiquitination via the Slimb F-box protein and proteasomes [43•,44•], suggesting that the processing reaction is indeed mediated by RUP.
Conclusions Rupture by RIP and RUP must now be added to the list of cellular tricks that regulate nuclear import and gene expression. Both mechanisms are well suited for a rapid and reversible activation of a gene expression program. Why nature has evolved two very different strategies for similar reactions is not clear in the moment, but future studies are expected to reveal the specific potencies of the two pathways. The examples discussed in this review also make it clear that RIP and RUP are not restricted to the activation of transcription factors — not even the regulation of nuclear import. In fact, cleaving off biologically active protein domains from precursors appears to be a very versatile biological concept. Both types of mechanisms, site-specific cleavage of membrane spans and the partial degradation of a protein by the proteasome, are fascinating strategies. Yet perhaps even more exciting is the question how regulatory circuits like the SREBP and the OLE pathway are controlled. Regulatory circuits typically require specific tactics to turn them on and to shut them down. It turns out that SREBP and SPT23 are extremely well suited for their biological tasks. In both cases the ER-bound precursors of these transcription factors receive their signals from their immediate environment, the ER-membrane. Cleavage of SREBP is induced by low cholesterol levels in the membrane, whereas SPT23 cleavage may be induced by membrane fluiditycontrolled interaction with the RSP5 ubiquitin ligase. Similarly, ER-bound ATF6, which controls the ER stress response (UPR), is presumably activated by unfolded proteins within the lumen of the ER. The idea that further cues, for example, signals transduced from sensors for temperature and cell growth, trigger SREBP or SPT23 processing is speculative. But how are the regulatory circuits turned off? First, cleavage of SREBP and SPT23 are directly repressed by high levels of cholesterol and UFAs, respectively. Second, the mature forms of SREBP, SPT23 and ATF6 transcription factors are very short lived. Indeed, both proteins appear to be degraded in the nucleus by proteasomes ([3,25•,32••]; M Rape, T Hoppe, S Jentsch, unpublished data). Although several details concerning these two regulation circuits have to await future studies, the two tales of SREBP and SPT23 make it amply clear that membrane-bound transcription factors are
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fabulously qualified for transmitting signals from the vicinity of a cellular membrane to the nucleus. Hence, it will be only a matter of time before we find out what other examples nature has in store.
Acknowledgements We thank Alexander Buchberger for discussions and comments.
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