Leucine-rich nuclear-export signals: born to be weak

Update

TRENDS in Cell Biology

Vol.15 No.3 March 2005

Research Focus

Leucine-rich nuclear-export signals: born to be weak Ulrike Kutay and Stephan Gu¨ttinger Swiss Federal Institute of Technology (ETH) Zu¨rich, Institute of Biochemistry, Schafmattstrasse 18, HPM F11.1, 8093 Zu¨rich, Switzerland

CRM1 mediates the nuclear export of proteins exposing leucine-rich nuclear-export signals (NESs). Most NESs bind to CRM1 with relatively low affinity. Recently, higheraffinity NESs were selected from a 15-mer random peptide library. Unexpectedly, complexes between high-affinity NESs and CRM1 accumulate at the cytoplasmic filaments of the nuclear pore complex (NPC). This finding suggests that high-affinity NES binding to CRM1 impairs the efficient release of export complexes from the NPC, explaining why leucine-rich NESs have evolved to be weak. Introduction Transport of macromolecules through nuclear pore complexes (NPCs) is mediated by soluble transport receptors, termed importins and exportins (or, collectively, karyopherins), that bind to specific signals present within their substrate molecules. The transport receptor–substrate interaction is regulated by Ran, a small GTPase required for the directionality of transport. Ran is concentrated in the nucleus, in which it is bound to GTP, whereas cytoplasmic Ran is in the GDP form. RanGTP controls substrate binding to importins and exportins in an opposing manner. Importins bind to their substrates in the cytoplasm and release them upon binding of nuclear RanGTP. Conversely, in the nucleus, exportins bind to their cargo cooperatively with RanGTP, resulting in the formation of stable export complexes. Export complexes are dissociated in the cytoplasm, concurrent with or after NPC passage, by a mechanism involving destabilization of export complexes by the Ran-binding domains (RBDs) of RanBP1 or Nup358/RanBP2 and hydrolysis of Ran-bound GTP promoted by RanGAP [1,2]. Exportins facilitate the translocation of a multitude of different proteins, ribonucleoprotein particles (RNPs) and RNAs through the NPC (Table 1). Of all characterized vertebrate exportins, the export receptor CRM1/Exportin1 has the broadest known substrate range. CRM1 is involved in nucleocytoplasmic exchange of various shuttling proteins such as transcription factors and cell cycle regulators, in nuclear export of RNP complexes and in nuclear exclusion of cytoplasmic proteins such as translation factors [3,4]. The majority of the export substrates of CRM1 contain a short, leucine-rich nuclear-export signal (NES) that was first identified in HIV Rev and protein kinase A inhibitor [5,6]. As is the case for other exportin–cargo interactions, NES binding to CRM1 is greatly stimulated by RanGTP [7]. However, in contrast to other characterized Corresponding author: Kutay, U. ([email protected]).

www.sciencedirect.com

exportin–substrate complexes, the affinity of most characterized NES substrates for CRM1–RanGTP is relatively low. Whereas exportins such as CAS [8], Exp-t [9] and Exp4 [10] bind to their substrates with affinities in the low-nanomolar range, most NES proteins display 100to 500-fold lower affinities for CRM1 [11,12]. Efficient binding of weak NESs to CRM1 in the nucleus was suggested to be stimulated by a CRM1-specific cofactor, RanBP3, a nuclear RanGTP-binding protein [13,14]. The reason why the CRM1–NES interaction differs from other exportin–cargo complexes has remained elusive. In a recent study, Engelsma et al. screened a random, 15-mer phage peptide library for strong CRM1-binding NESs [15]. Indeed, high-affinity peptide interactors of CRM1 were identified. These artificial NES sequences resemble natural NESs but turn out to be too strong to be optimal in vivo. Engelsma et al. showed that these highaffinity NESs are trapped in a complex with CRM1 at the NPC when overexpressed in living cells. These findings not only reveal a new, terminal NPC binding site for CRM1 but also help to explain why natural NESs have maintained a low affinity for CRM1 – to enable clearance of the export receptor from the NPC. Selecting strong NESs Leucine-rich NESs conform more or less to the consensus F-x2–3-F-x2–3-F-x-F (FZL,I,V,F,M; x is any amino acid) [3,16]. A wide variety of functional NES sequences have been identified, and their diversity is relatively high (Figure 1a). The presence of regularly spaced, large hydrophobic amino acids such as leucine or isoleucine seems to be the most important feature of the signal. The intervening amino acids are often negatively charged, polar or small [3,16]. Moreover, the protein context is important for NES activity. For example, the isolated NES of HIV Rev binds to CRM1 much more weakly than does the fulllength Rev protein [12]. This implies that an NES might Table 1. Mammalian exportinsa Exportin CRM1/Exp1 CAS Exp-t/Xpo-t Exp4 Exp5 Exp6 Exp7 Imp13b a

Export cargo proteins containing leucine-rich NESs, snurportin 1 Importin-a tRNAs eIF5A Pre-miRNAs, tRNAs, minihelix RNAs, eEF1A, JAZ Profilin–actin p50RhoGAP, 14–3–3 sigma eIF1A

For further details, refer to the Reference list [1,2,24–26]. Note that the main function of Imp13 is in nuclear import [27].

b

Update

122

(a)

(b)

TRENDS in Cell Biology

Protein

NES sequence

HIV-1 Rev MVM NS2 PKI MAPKK NMD3 An3 IκBα Cyclin B1 TFIIIA

L-PPL-ERLTL MTKKF-GTLTI LALKL-AGLDI LQKKL-EELEL LAEML-EDLHI LDQQF-AGLDL MVKEL-QEIRL LCQAF-SDVIL L-PVL-ENLTL

NES consensus

Φx2-3Φx 2-3ΦxΦ

Peptide

NES sequence

NE localization

S0 S1

L-ARLFSALGV L-ARLFSALSV

+

P0 P2

L-SSLFSGFSV L-SSLFSGLSV

RanBP1 wt NES RanBP1 mut NES

VAEKL-EALSV LAELF-EALSV

+ +

Consensus

Lx2-LF-x2-LSV TRENDS in Cell Biology

Figure 1. NES features and examples. (a) Examples of viral and vertebrate leucinerich NESs [3,15]. Conserved hydrophobic residues are in red. (b) S0 and P0 NES-like peptides identified in the random peptide library screen, their variants and the RanBP1 NES peptides, as used by Engelsma et al., are depicted [15]. Mutations in the individual peptide sequences are highlighted in boxes. ‘NE localization’ refers the accumulation of NES peptide–GFP fusions at the nuclear rim, as observed in [15].

require an appropriate context to adopt the conformation needed for CRM1 binding, and that flanking residues might also contribute to NES affinity. Even though the binding affinities for CRM1 vary between different NESs, they are generally low. What exactly determines the strength of interaction with CRM1 has long remained unclear. To isolate strong NESs and to gain insight into NES diversity, Engelsma et al. searched for CRM1-binding peptides by affinity selection on immobilized CRM1 using phage display. Selection was performed either in the absence or presence of RanGTP. Each selection highly enriched a unique peptide, termed S0 and P0, respectively. Both peptides conform to the NES consensus and, surprisingly, are much alike (Figure 1b). Green fluorescent protein (GFP) fusions to these peptides were efficiently exported to the cytoplasm in vivo. By changing the penultimate amino acid in the S0 peptide – from an unusual glycine at this position to serine – they created an NES-like peptide, termed S1, harboring a low nanomolar affinity for CRM1. Binding of the S1 peptide to CRM1 is sufficiently strong that it occurs even in the absence of RanGTP. Further permutations of the S and P peptides identified the sequence LxxLFxxLSV as optimal for highaffinity interaction with CRM1 (Figure 1b).

‘Supraphysiological’ NESs reveal novel NPC binding site for CRM1 CRM1-mediated translocation through the NPC is energy-independent and believed to occur by facilitated diffusion, relying on multiple low-affinity interactions with www.sciencedirect.com

Vol.15 No.3 March 2005

phenylalanine glycine (FG)-repeat-containing nucleoporins. After transfer through the NPC, export complexes must be disassembled. Termination of CRM1-mediated export has long been thought to involve Nup214, which is located at the cytoplasmic ring of the NPC. CRM1 binds strongly to Nup214, in a RanGTP- and NES-dependent manner [11,17,18]. Nup214 lies in close proximity to the cytoplasmic NPC filaments, consisting of Nup358–RanBP2, which combines all of the features required for the efficient disassembly of export complexes. Nup358 possesses FG repeat motifs for transport-receptor binding, in addition to four RanBP1-type RanGTP-binding domains, and it is stably associated with sumoylated RanGAP [19,20]. The RanBP1-like domains of Nup358 are predicted to dissociate RanGTP from export complexes and to present it to RanGAP, resulting in export-complex disassembly. It has remained unclear if CRM1-containing export complexes are indeed transferred from Nup214 to Nup358 for disassembly. Nup358 and CRM1 are known to associate but independently of bound cargo. This interaction has therefore been suggested to have a role after export-complex disassembly, that is, in the recycling of empty CRM1 to the nucleus [21,22]. The strong artificial NESs identified by Fornerod and colleagues have enabled discovery of a novel, terminal CRM1-binding site at Nup358 (Figure 2). When overexpressed, an S1–GFP fusion protein strongly accumulated at the nuclear rim, precisely at the position of the cytoplasmic NPC filaments. This localization was concluded to be dependent on Nup358 because depletion of Nup358, but not of Nup214, by RNAi removed S1–GFP from the nuclear envelope (NE). Moreover, S1 peptides were able to retrieve Nup358 from Xenopus egg extracts. The interaction between S1–GFP and Nup358 at the NPC was shown to be mediated by CRM1 because binding was sensitive to the CRM1 inhibitor leptomycin B (LMB), and S1–GFP accumulation at the NPC correlated with increased amounts of CRM1 at the NE. However, RanGTP did not accumulate at the NPC as part of the CRM1–S1–GFP complex, consistent with the observation that Ran could still be removed from trimeric CRM1–S1–RanGTP complexes in the presence of RanBP1 in vitro. Altogether, these experiments reveal a second, cargo-dependent CRM1-binding site at Nup358. Concluding remarks In summary, the data presented by Fornerod and colleagues are consistent with the model that high-affinity NES binding to CRM1 impairs the efficient release of CRM1–NES export complexes from the NPC. To strengthen their model, Engelsma et al. changed the weak NES of RanBP1 into one fitting the strong NES consensus (Figure 1b). Similar to the S1 NES, the mutated RanBP1 NES promoted NE localization. In conclusion, Nup358 is suggested to function as the terminal translocation site for CRM1 export complexes, serving their disassembly at the cytoplasmic filaments of the NPC (Figure 2). Inefficient disassembly of CRM1–NES export complexes might have dominant negative effects on CRM1mediated export in general. Interestingly, overexpressed S1–GFP sequestered CRM1 at the nuclear rim and in the

Update

TRENDS in Cell Biology

(a)

RanGDP

Vol.15 No.3 March 2005

123

(b)

RanGDP Nup358 RBDs RanGAP Cytoplasm Zn2+-finger domain Nucleus Nup88/Nup214 CRM1

RanGTP

NES substrate TRENDS in Cell Biology

Figure 2. CRM1-mediated NES protein export. (a) The CRM1 transport cycle. In the nucleus, RanGTP stimulates binding of CRM1 to NES substrates. After passage through the NPC, the CRM1–RanGTP–NES substrate complex is disassembled at the cytoplasmic filaments by the concerted action of RanGAP and RanBP1 or RanBP1-like domains of Nup358–RanBP2. The NES substrate is released to the cytoplasm and empty CRM1 is recycled back to the nucleus. (b) Model of CRM1 export complex disassembly. After passage through the central NPC channel, the CRM1–RanGTP–NES complex is transferred to the cargo-dependent CRM1-binding site of Nup358–RanBP2 (possibly after passing Nup214). The binding of CRM1 to this site is LMB sensitive and is highly stimulated by the binding of strong NESs. Export-complex disassembly is expected to occur here, stimulated by the RanBP domains of Nup358- and Nup358-associated RanGAP. CRM1 is released into the cytoplasm and, for recycling into the nucleus, binds to a different, cargo-independent CRM1-binding site of Nup358. For clarity, export complexes and recycling CRM1 are shown on separate NPC filaments.

cytoplasm, indicating that complex formation with highaffinity substrates might hinder the recycling of CRM1 to the nucleus. It remains to be seen whether occupancy of the Nup358 disassembly site by CRM1–NES complexes has dominant negative effects on other import or export pathways. The results of these studies will show whether the S1 peptide has a future as a novel inhibitor of the CRM1 pathway. The disassembly of other exportin–cargo complexes at the NPC has not been studied in great detail. Exportin-t, which has a high affinity for its export cargo tRNA, also binds to Nup358 in vitro [23] but is not localized to the nuclear rim at steady state [9], suggesting that dissociation of this export complex from the NPC is efficient in vivo. Clearly, NES binding modulates the NPC-binding strength of CRM1 and changes its steady-state localization. It is not yet known how CRM1 interacts with nucleoporins and NES cargos; this discovery awaits the structural characterization of these complexes – a challenging task, for which the S1 peptide might be a useful reagent. Taken together, these data suggest that natural NESs are of relatively low affinity to avoid defects in exportcomplex disassembly. It should be noted that there are natural CRM1 substrates that bind to CRM1 with high affinity. One example is snurportin, a factor involved in small nuclear ribonucleoprotein import that uses the CRM1 export pathway for recycling [12]. Snurportin does not contain a canonical NES-like sequence, and binds to CRM1 through a larger domain. The high-affinity interaction between snurportin and CRM1 is presumably tolerated because dissociation of the complex from the NPC is efficient. This might be explained by the different mode of snurportin binding to CRM1 or because dissociation is assisted in some other way. Moreover, expression levels of snurportin are likely to be low compared with the www.sciencedirect.com

overexpressed S1–GFP protein. Further exceptions might exist, such as the NES of parvovirus minute virus of mice NS2 protein, which does not fit the suggested NES consensus (Figure 1a,b) but binds strongly to CRM1. Another example is the NES in human NMD3, which resembles the strong NES consensus (Figure 1a). NMD3 has been suggested to function as export adaptor for 60S preribosomal subunits [1]. Perhaps strong NESs are required to facilitate the export of large particles because large complexes supposedly transit the NPC more slowly than small exportin–substrate complexes. At the final stages of export, additional factors might aid disassembly of larger, more stable export complexes. Despite the fact that many NES proteins have a low affinity for CRM1 in vitro, they are exported efficiently in vivo. Thus, mechanisms must have evolved to guarantee export-complex formation at a reasonable rate. Cofactors such as RanBP3 are thought to facilitate NES substrate recognition in vivo [13,14]. Local restriction of NES complex formation might also contribute to efficient NES binding to CRM1 in the nucleus. Acknowledgements We acknowledge funding from the Swiss National Science Foundation, and we thank Howard Fried and Ivo Zemp for critical reading of the manuscript. We apologize to our colleagues for not citing all original publications owing to space limitations.

References 1 Fried, H. and Kutay, U. (2003) Nucleocytoplasmic transport: taking an inventory. Cell. Mol. Life Sci. 60, 1659–1688 2 Mosammaparast, N. and Pemberton, L.F. (2004) Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends Cell Biol. 14, 547–556 3 Fornerod, M. and Ohno, M. (2002) Exportin-mediated nuclear export of proteins and ribonucleoproteins. Results Probl. Cell Differ. 35, 67–91

Update

124

TRENDS in Cell Biology

4 Bohnsack, M.T. et al. (2002) Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm. EMBO J. 21, 6205–6215 5 Fischer, U. et al. (1995) The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475–483 6 Wen, W. et al. (1995) Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463–473 7 Fornerod, M. et al. (1997) CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90, 1051–1060 8 Kutay, U. et al. (1997) Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. Cell 90, 1061–1071 9 Kutay, U. et al. (1998) Identification of a tRNA-specific nuclear export receptor. Mol. Cell 1, 359–369 10 Lipowsky, G. et al. (2000) Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes. EMBO J. 19, 4362–4371 11 Askjaer, P. et al. (1999) RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase. Mol. Cell. Biol. 19, 6276–6285 12 Paraskeva, E. et al. (1999) CRM1-mediated recycling of snurportin 1 to the cytoplasm. J. Cell Biol. 145, 255–264 13 Englmeier, L. et al. (2001) RanBP3 influences interactions between CRM1 and its nuclear protein export substrates. EMBO Rep. 2, 926–932 14 Lindsay, M.E. et al. (2001) Ran-binding protein 3 is a cofactor for Crm1-mediated nuclear protein export. J. Cell Biol. 153, 1391–1402 15 Engelsma, D. et al. (2004) Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358. EMBO J. 23, 3643–3652 16 La Cour, T. et al. (2004) Analysis and prediction of leucine-rich nuclear export signals. Protein Eng. Des. Sel. 17, 527–536 17 Fornerod, M. et al. (1997) The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. EMBO J. 16, 807–816

Vol.15 No.3 March 2005

18 Kehlenbach, R.H. et al. (1999) A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export. J. Cell Biol. 145, 645–657 19 Matunis, M.J. et al. (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135, 1457–1470 20 Mahajan, R. et al. (1997) A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97–107 21 Singh, B.B. et al. (1999) The zinc finger cluster domain of RanBP2 is a specific docking site for the nuclear export factor, exportin-1. J. Biol. Chem. 274, 37370–37378 22 Bernad, R. et al. (2004) Nup358/RanBP2 attaches to the nuclear pore complex via association with Nup88 and Nup214/CAN and plays a supporting role in CRM1-mediated nuclear protein export. Mol. Cell. Biol. 24, 2373–2384 23 Kuersten, S. et al. (2002) Steady-state nuclear localization of exportin-t involves RanGTP binding and two distinct nuclear pore complex interaction domains. Mol. Cell. Biol. 22, 5708–5720 24 Mingot, J.M. et al. (2004) Exportin 7 defines a novel general nuclear export pathway. EMBO J. 23, 3227–3236 25 Kim, V.N. (2004) MicroRNA precursors in motion: exportin-5 mediates their nuclear export. Trends Cell Biol. 14, 156–159 26 Chen, T. et al. (2004) Nucleocytoplasmic shuttling of JAZ, a new cargo protein for exportin-5. Mol. Cell. Biol. 24, 6608–6619 27 Mingot, J.M. et al. (2001) Importin 13: a novel mediator of nuclear import and export. EMBO J. 20, 3685–3694

0962-8924/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2005.01.005

Vacuolar proteases livening up programmed cell death Eric Lam Biotech Center, Rutgers University, 59 Dudley Road, Foran Hall, New Brunswick, NJ 08901, USA

The molecular identity of the key executioners involved in controlling plant programmed cell death (PCD) has been elusive. In a recent paper published in Science, Hatsugai and coworkers reported that a well-characterized protease called VPE from the plant cell vacuole can cleave caspase-specific substrates and is required for cell death activation by tobacco mosaic virus. This work provides clear evidence for the importance of the vacuole in plant PCD and a novel regulatory function for this organelle as well as for VPE proteases. Programmed cell death (PCD) takes many shapes and forms and is recognized to be ubiquitous among living cells ranging from prokaryotes to eukaryotes [1–3]. Its origin(s) is still uncertain, but it clearly plays crucial roles in organized development and stress survival of multicellular organisms. In the case of animal cell apoptosis, which is the most well-characterized form of PCD that ultimately results in the engulfment and removal of the Corresponding author: Lam, E. ([email protected]). Available online 2 February 2005 www.sciencedirect.com

dying cell by its neighbours, a combination of molecular and genetic approaches has revealed the basic chain of regulators and executioners that are responsible for and dedicated to carrying out the organized and systematic cell suicide. A prominent point of control in apoptosis is a family of specialized cysteine proteases called caspases (cysteine-containing aspartate-specific proteases) that functions as the integration point for life-or-death decision of the cell. Controlling the activity of caspases is often viewed as a crucial switch for diverse pathways in the cell to influence PCD under stress or during development in animals [1]. In spite of the long-recognized existence of PCD in plants [4], the molecular description of a central executioner has been elusive. This ignorance has impeded the progress of PCD research over the past decade and makes the integration of results from diverse plant cell death models difficult. However, the recent work from Hara-Nishimura’s laboratory [5] might now provide a first link between the plant legumain called vacuolar processing enzyme (VPE) and caspase-like functions. In addition to suggesting a new role for this vacuole-localized