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4 Bohnsack, M.T. et al. (2002) Exp5 exports eEF1Avia tRNA from nucleiand synergizes with other transport pathways to confine translation tothe cytoplasm. EMBO J. 21, 6205–6215

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7 Fornerod, M. et al. (1997) CRM1 is an export receptor for leucine-richnuclear export signals. Cell 90, 1051–1060

8 Kutay, U. et al. (1997) Export of importin alpha from the nucleus ismediated by a specific nuclear transport factor. Cell 90, 1061–1071

9 Kutay, U. et al. (1998) Identification of a tRNA-specific nuclear exportreceptor. Mol. Cell 1, 359–369

10 Lipowsky, G. et al. (2000) Exportin 4: a mediator of a novel nuclearexport pathway in higher eukaryotes. EMBO J. 19, 4362–4371

11 Askjaer, P. et al. (1999) RanGTP-regulated interactions of CRM1 withnucleoporins and a shuttling DEAD-box helicase. Mol. Cell. Biol. 19,6276–6285

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13 Englmeier, L. et al. (2001) RanBP3 influences interactions betweenCRM1and its nuclear protein export substrates.EMBORep. 2, 926–932

14 Lindsay, M.E. et al. (2001) Ran-binding protein 3 is a cofactor forCrm1-mediated nuclear protein export. J. Cell Biol. 153, 1391–1402

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17 Fornerod, M. et al. (1997) The human homologue of yeast CRM1 is in adynamic subcomplex with CAN/Nup214 and a novel nuclear porecomponent Nup88. EMBO J. 16, 807–816

Corresponding author: Lam, E. (Lam@aesop.rutgers.edu).Available online 2 February 2005

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18 Kehlenbach, R.H. et al. (1999) A role for RanBP1 in the release ofCRM1 from the nuclear pore complex in a terminal step of nuclearexport. J. Cell Biol. 145, 645–657

19 Matunis, M.J. et al. (1996) A novel ubiquitin-like modificationmodulates the partitioning of the Ran-GTPase-activating proteinRanGAP1 between the cytosol and the nuclear pore complex. J. CellBiol. 135, 1457–1470

20 Mahajan, R. et al. (1997) A small ubiquitin-related polypeptideinvolved in targeting RanGAP1 to nuclear pore complex proteinRanBP2. Cell 88, 97–107

21 Singh, B.B. et al. (1999) The zinc finger cluster domain of RanBP2 is aspecific 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 porecomplex via association with Nup88 and Nup214/CAN and plays asupporting role in CRM1-mediated nuclear protein export. Mol. Cell.Biol. 24, 2373–2384

23 Kuersten, S. et al. (2002) Steady-state nuclear localization ofexportin-t involves RanGTP binding and two distinct nuclear porecomplex interaction domains. Mol. Cell. Biol. 22, 5708–5720

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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

incontrollingplantprogrammedcell death (PCD)hasbeen

elusive. In a recent paper published in Science, Hatsugai

and coworkers reported that a well-characterized pro-

tease 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 andforms and is recognized to be ubiquitous among living cellsranging from prokaryotes to eukaryotes [1–3]. Its origin(s)is still uncertain, but it clearly plays crucial roles inorganized development and stress survival of multi-cellular organisms. In the case of animal cell apoptosis,which is the most well-characterized form of PCD thatultimately results in the engulfment and removal of the

dying cell by its neighbours, a combination of molecularand genetic approaches has revealed the basic chain ofregulators and executioners that are responsible for anddedicated to carrying out the organized and systematiccell suicide. A prominent point of control in apoptosis is afamily of specialized cysteine proteases called caspases(cysteine-containing aspartate-specific proteases) thatfunctions as the integration point for life-or-death decisionof the cell. Controlling the activity of caspases is oftenviewed as a crucial switch for diverse pathways in the cellto influence PCD under stress or during development inanimals [1]. In spite of the long-recognized existence ofPCD in plants [4], the molecular description of a centralexecutioner has been elusive. This ignorance has impededthe progress of PCD research over the past decade andmakes the integration of results from diverse plant celldeath models difficult. However, the recent work fromHara-Nishimura’s laboratory [5] might now provide a firstlink between the plant legumain called vacuolar process-ing enzyme (VPE) and caspase-like functions. In additionto suggesting a new role for this vacuole-localized

Update TRENDS in Cell Biology Vol.15 No.3 March 2005 125

protease, this discovery points to the possibility that ananalogous pathway for PCD activationmight be conservedin animal cells as well.

Proteolytic switches as PCD regulators in plants

In addition to apoptotic cell death, animal cells can also diethrough autophagic morphotypes that do not end withengulfment. Interestingly, it appears that caspases couldalso play important roles in these types of PCD pathways[1,2]. With their inherent cell wall, which precludes a‘clean’ death by engulfment, cell death in plant cells andyeasts might have more similarities with these types ofanimal cell death pathways [2,3]. A prominent organellein plant cells and yeasts is the vacuole, which in matureplant cells can take up the majority of the volume of thecell. Vacuoles are known to play an important storagefunction in seeds as well as serving as a reservoir forhydrolytic enzymes such as proteases and nucleases invegetative tissues [6,7]. They also play a major role in therecycling of cellular contents through pathways such asautophagy during nutrient deficiency in both yeasts andplants [8]. Lysosomes in animals serve analogous func-tions and, along with vacuoles in plants and yeasts, arerecognized as likely candidates serving key regulatoryfunctions in non-apoptotic cell deaths [1,3]. However, theregulatory mechanism and sequence of events in this typeof cell death pathway remain unclear.

Since 1998, the involvement of caspase-like pro-teases (CLPs) to control PCD activation in plants hasbeen suggested from pharmacological studies by differ-ent laboratories using various cell death models [9,10].Although these results implicated the crucial role ofproteases in regulating plant PCD, interpretation ofthese results has been controversial because themolecular identity of the protease(s) involved remainedelusive.

CLP activity of the legumain VPE and its role in plant cell

death

Using the hypersensitive response (HR) cell death inducedby tobacco mosaic virus (TMV) as a model system (Box 1),Hatsugai et al. have recently reported the identification ofthe vegetative form of the VPE as the CLP induced duringthe onset of the HR [5]. This was first indicated by affinitylabeling studies and was then subsequently confirmedby immunodepletion with antibodies against VPE. In

Box 1. Hypersensitive response: a model of induced plant PCD

Upon challenge with incompatible pathogens, plants can mount a

rapid immunity response that typically involves rapid cell death – the

hypersensitive response (HR) – at the site of initial pathogen ingress

[20]. Coordinate with the macroscopic tissue collapse that is easily

visible within 12 to 30 h of pathogen inoculation, depending on the

particular host–pathogen interaction, a series of defence responses

including cell wall thickening and defence-related gene expression is

activated to suppress proliferation of the invading pathogen as well as

to provide broad-range resistance to subsequent challenge by

different types of pathogens. Salicylic acid and nitric oxide are

recognized as signaling molecules in both local and systemic events

during the HR, and their balance could control activation of cell death

in plant cells. In a gene-for-gene system for host–pathogen interaction,

incompatibility is determined by the presence of an avirulence gene in

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addition, VPE activity in leaf extracts was inhibited bycaspase inhibitors such as YVAD-CHO (for caspase-1) orxVAD-fmk (a general caspase inhibitor), but not a caspase-3-specific inhibitor such as DEVD-CHO, a serine proteaseinhibitor such as PMSF, or a papain-type-proteaseinhibitor such as E-64. This confirmed and extended theprevious observation that the legumain VPE has alterna-tive usage of aspartate or asparagines at the substratecleavage site [11]. It is also consistent with the assignmentof legumains and caspases to the same clan of proteasefamily based on the structural conservation of theircatalytic dyad [12]. Importantly, Hatsugai et al. showedthat VPEs are essential for PCD activation duringinduction of the HR. Using a virus-induced gene silencing(VIGS) strategy, four independent lines of transgenictobacco with reduced VPE expression were generated. Inaddition to a concomitant loss in detectable activation ofVPE and caspase-1 substrate cleavage activities upon HRinduction as mimicked by salicylic acid treatment, thesesuppressed lines also failed to show any signs of PCD asassayed by chromatin fragmentation or visual tissuecollapse when challenged with TMV. Although defencegene activation was not reduced, virus proliferation ismarkedly increased in the VPE-suppressed plants. Theseobservations showed strong molecular evidence for thecausal relationship between VPE and the observed CLPactivity. Furthermore, these observations support the ideathat HR cell death is an important component for virusrestriction and could be uncoupled from defence geneactivation [3].

How might VPE be involved in HR cell death activation?

Although VPE is expressed before PCD activation, it isimportant to note that VPE and its associated CLPactivities can be further increased by salicylic acidtreatment, the signaling molecule associated with plantdefence (Box 1). In addition, expression of VPE at thetranscript level is also upregulated transiently in the earlyphase of HR activation. Thus, one component of VPEactivation appears to be at the level of VPE transcription.As an early sensor for PCD activation, such as the initiatorclass of caspases [1], regulation of VPE at the posttransla-tional level through protein–protein interaction would bean interesting possibility for future examination.

VPEs are known to localize to the vacuole, and they areresponsible for the proteolytic processing of other vacuolar

the pathogen and a corresponding resistance gene in the plant host

[21]. Although the role of cell death in disease resistance is uncertain

for some pathogens, such as bacteria, HR cell death likely functions to

restrict the systemic movement of plant viruses within the host plant.

For the TMV–tobacco interaction, HR induction is dependent on the

presence of the N gene in the host, and the signaling process is

temperature sensitive. Thus, TMV can be allowed to multiply

systemically in plant host cultivars containing the N gene by keeping

the plants at 30 8C. Upon transfer to the permissive temperature of

25 8C, HR cell death can be activated in a semi-synchronized fashion in

the infected plant. The recent work of Hatsugai et al. [5] uses this

genetically defined programmed cell death (PCD) system for the

elucidation of the unexpected role that vacuolar processing enzymes

(VPEs) might play in the HR.

Update TRENDS in Cell Biology Vol.15 No.3 March 2005126

proteins such as lectins, a-amylase inhibitor and carboxy-peptidase Y [6,7,12]. Although the VPE target(s) thatmediates HR cell death is unknown, Hatsugai et al.nevertheless observed a dramatic inhibition of vacuolecollapse in VPE-suppressed plants. This observationsuggests that disruption of vacuole integrity is dependenton VPEs, and this step might be crucial for at least someforms of HR cell death, as observed in the case of thedevelopmental cell death during maturation of trachearyelements [13].

Genetic complexities of plant PCD pathways and

regulators

Like all exciting observations, the results of Hatsugaiet al. (5) raise many new questions. One of the immediatepoints of interest is the identity of the targets of VPEs thatpresumably are regulated (activation or suppression) bythis protease and are important for HR cell deathinduction. This includes the mechanism through whichVPE activation might lead to vacuole collapse. Also, themechanism through which the vacuole-localized VPEcould exert its control on cell death events that ultimatelyleads to systematic destruction of chromatin in thenucleus and dismantling of the cytoplasm would nodoubt provide new appreciation of intracellular crosstalkbetween organelles. Whether vacuole collapse is a neces-sary step for activation of HR cell death or whether theseare two parallel cascades of events that follow after VPEactivation is an important issue to resolve.

How many CLPs are there in plants? Using thesame HR system as employed in the work of Hatsugaiet al. [5], Chichkova et al. [14] detected a CLP activitythat appeared upon TMV-induced PCD that has thespecificity of cleaving TATD and GEQD at the substratetarget. Biotin–TATD–CHO, but not Biotin–DEVD–CHO(a Caspase-3-specific inhibitor), delayed HR cell death butnot defence gene activation. Of particular interest in thisstudy is their use of an in vivo nuclear-localized proteasedetector that indicated this CLP activity might belocalized in the nucleus. This work suggests that a CLPactivity distinct from VPE could also be activated duringTMV-induced HR. In both studies, a Caspase-3 inhibitorfailed to suppress the observed cell death or the associatedCLP activity. By contrast, a previous study with bacteria-induced HR in tobacco [15] and a more recent study withoat cell death induced by the fungal toxin victorin [16]suggested the presence of a Caspase-3-like CLP in thissystem. In addition, the latter study biochemicallyseparated two CLP activities from victorin-treated oatleaves that cleave either a Caspase-3-specific substrate(zDEVD–AFC) or a more general caspase target substrate(zVAD–AFC). The zVAD-specific CLP activity was resolvedinto two related proteins both containing subtilisin-likeserine protease motifs and shows partial sensitivity toPMSF and E-64 inhibitors. These novel proteases havesubsequently been named ‘saspases’ (for serine-containingaspartate-specific proteases). Although their functionalsignificance in relationship to the associated PCD remainsto be defined, it is interesting to note that saspases arerapidly secreted into the extracellular fluids upon victorintreatment. These studies thus point to the presence of

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multiple CLPs that could be localized to differentsubcellular compartments upon PCD induction. In thecase of VPE and the TATDase activities induced duringTMV-activated HR, the proteases involved are cysteineproteases, and thus they would fit the functional descrip-tion of CLPs, although they might not share anysignificant sequence homologies with classical caspases.For saspases, however, the similarity with caspases wouldbe solely at the level of target specificity, and thus theywould not be considered as bona fide CLPs.

Finally, complete abolishment of all VPE-encoded geneswas recently achieved in Arabidopsis thaliana by mutantstacking [17], and, aside from the expected seed proteinprocessing defects, the resultant quadruple mutants donot show any obvious phenotypes with respect to seedgermination, vegetative characteristics and senescence.These interesting observations suggest that develop-mental PCD, such as tracheid formation, can occur in arelatively normal manner in the absence of VPEs. Inaddition, a loss-of-functionmutant of VPEg inArabidopsisresulted in partial suppression of CLP activities that areinduced upon infection with an avirulent bacterium [18].This is correlated with increased susceptibility of vpegplants to infection by bacterial, fungal and viral patho-gens. However, the effect of vpeg mutation on HR celldeath was not dramatic [18], and whether the other threeArabidopsis VPE-encoding genes might have functionsoverlapping those of VPEg would need to be addressedwith the quadruple mutant [17].

Concluding remarks

Although vacuole collapse has been shown to be a keyevent for the cell death crucial in the formation of vasculartissue [13], VPEs are apparently dispensable for this andother essential PCD pathways in planta. Further analysesusing vacuole-related mutants, and the molecular handlethat the VPE-encoding genes provide, should generateexciting information in the near future on novel mechan-isms of PCD control in plants. With the recent discoveryof legumain-like proteins in the lysosome of animal cells[12,19], these findings should fuel research in the study ofanimal PCD and provide answers to possible origins of celldeath pathways in eukaryotes.

AcknowledgementsThe support by the USDA to plant cell death research in the Lamlaboratory at Rutgers University and critical reading of themanuscript byNaohide Watanabe is gratefully acknowledged.

References

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5 Hatsugai, N. et al. (2004) A plant vacuolar protease, VPE, mediatesvirus-induced hypersensitive cell death. Science 305, 855–858

6 Rojo, E. et al. (2003) A unique mechanism for protein processing anddegradation in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A.100, 7389–7394

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10 Watanabe, N. and Lam, E. (2004) Recent advance in the study ofcaspase-like protease and Bax inhibitor-1 in plants: their possibleroles as regulators of programmed cell death. Mol. Plant Pathol.5, 65–70

11 Hiraiwa, N. et al. (1999) Vacuolar processing enzyme is self-catalytically activated by sequential removal of the C-terminal andN-terminal propeptides. FEBS Lett. 447, 213–216

12 Chen, J-M. et al. (1998) Identification of the active site of legumainlinks it to caspases, clostripain and gingipains in a new clan ofcysteine endopeptidases. FEBS Lett. 441, 361–365

13 Obara, K. et al. (2001) Direct evidence of active and rapid nucleardegradation triggered by vacuole rupture during programmed celldeath in Zinnia. Plant Physiol. 125, 615–626

14 Chichkova, N.V. et al. (2004) A plant caspase-like protease activatedduring the hypersensitive response. Plant Cell 16, 157–171

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15 del Pozo, O. and Lam, E. (1998) Caspases and programmed cell deathin the hypersensitive response of plants to pathogens. Curr. Biol. 8,1129–1132

16 Coffeen, W.C. and Wolpert, T.J. (2004) Purification and characteriz-ation of serine proteases that exhibit caspase-like activity and areassociated with programmed cell death in Avena sativa. Plant Cell 16,857–873

17 Gruis, D. et al. (2004) Storage protein accumulation in the absence ofthe vacuolar processing enzyme family of cysteine proteases. PlantCell 16, 270–290

18 Rojo et al. (2004) VPEg exhibits a caspase-like activity thatcontributes to defense against pathogens. Curr. Biol. 14, 1897–1906

19 Li, D.N. et al. (2003) Multistep autoactivation of asparaginylendopeptidase in vitro and in vivo. J. Biol. Chem. 278, 38980–38990

20 Lam, E. et al. (2001) Programmed cell death, mitochondria and theplant hypersensitive response. Nature 411, 848–853

21 Dangl, J.L. and Jones, J.D.G. (2001) Plant pathogens and integrateddefence responses to infection. Nature 411, 826–833

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