Alternative nuclear transport forcellular protein quality controlApril Rose and Christian Schlieker

Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, P.O. Box 208114, Bass 236A,

New Haven, CT 06520-8114, USA

Opinion

Glossary

Autophagy: cellular degradation pathway that degrades large structures such

as aggregated proteins and organelles by engulfing the structure in an

isolation membrane and targeting it to the lysosome for degradation.

Isolation membrane: double-membrane structure that elongates to engulf

proteins or organelles destined to be degraded by the lysosome.

Laminopathies: designation for a large number of diseases such as Emery–

Dreifuss muscular dystrophy and progeria caused by mutations in nuclear

lamins and proteins associated with the lamina.

Lysosome: cellular organelle of low pH that is capable of degrading cellular

components regardless of size.

Multivesicular body (MVB): endosome containing numerous vesicles formed

by endosomal sorting complex required for transport (ESCRT) proteins

containing proteins to be degraded. MVBs eventually fuse with the lysosome

and the vesicles with their cargo are degraded.

Nuclear egress: the process by which herpesvirus capsids are transported from

the nucleus to the cytoplasm. This requires vesiculation of the INM and

formation of a vesicular intermediate in the PNS that fuses with the ONM to

allow naked capsids access to the cytoplasm.

Primary envelopment: acquisition by HSV-1 of a membrane envelope derived

from the INM on nuclear egress. This envelope is lost on fusion with the ONM.

Ribonucleoprotein (RNP) granule: dense RNP structure comprising RNA and

protein shown to traffic from the nucleus to the cytoplasm via a membrane

budding pathway that takes it through the NE.

Secondary envelopment: acquisition by HSV-1 of a double-membrane

envelope. The interior membrane is the final viral membrane envelope that

includes glycoproteins required for subsequent host cell entry; the exterior

membrane of this structure will fuse with the plasma membrane to release the

Herpesvirus capsids traverse the nuclear envelope (NE)by utilizing an unusual export pathway termed nuclearegress. In this process, the viral capsid is delivered intothe perinuclear space (PNS), producing a vesicular inter-mediate after fission. After fusion with the outer nuclearmembrane (ONM), the naked capsid is released into thecytosol. A recent study now suggests that this pathwaymight be an endogenous cellular pathway, co-opted byviruses, that serves to transport cellular cargo exceedingthe size limit imposed by the nuclear pore complex(NPC). We propose that one function of this pathwayis to transport nuclear protein aggregates to the cyto-solic autophagy machinery. Our model has implicationsfor our understanding of laminopathies and related dis-eases affecting proteins residing at the inner nuclearmembrane (INM) and nuclear lamina.

Nuclear egress: a transport pathway for viral capsidsIn the late phase of the lytic cycle of a productive herpes-virus infection, the DNA genomes of progeny viruses arepackaged into viral capsids in the host cell nucleus. Thecompletely assembled nucleocapsid has a diameter of ap-proximately 125 nm and although this number variesbetween different members of the herpesvirus family [1],the dimensions of the nucleocapsid greatly exceed the39 nm size limit of the NPC [2]. For this reason, nuclearexport of capsids cannot rely on the canonical machinerythat utilizes the NPC as a conduit to the cytosol. Instead,most if not all herpesviruses follow a pathway termednuclear egress [3] (see Glossary). In the first step of nuclearegress, designated primary envelopment, the capsidapposes to the INM of the NE and buds into the PNS toproduce a vesicular intermediate (Figure 1a). On fusion ofthis intermediate with the ONM, the naked capsid isreleased into the cytosol and has thus crossed the twomembranes that define the borders of the NE. What followsis a process termed secondary envelopment, in which thecapsid is enveloped by a double membrane (Figure 1a).From a topological perspective, we consider this latterprocess highly reminiscent of isolation membrane forma-tion [4] at the onset of autophagy. Finally, the doublemembrane closes. Following fusion of the ONM with theplasma membrane, the mature virion is then released fromthe cell.

Corresponding author: Schlieker, C. (christian.schlieker@yale.edu).Keywords: Torsin A; LINC complex; chaperones; nuclear envelope; egress of nuclearaggregates.premature aging.

0962-8924/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.101

A large body of work [3,5] has established the individualsteps that underlie nuclear egress. Initially, the nuclearlamina is dissolved in a process that requires phosphory-lation of lamins and other INM-resident proteins, includ-ing Emerin, by recruitment of cellular kinases and viavirus-encoded kinases [6–8]. The capsid is then recruitedto the INM via the nuclear egress complex, which isconserved in all herpesvirus subfamilies. For human her-pesvirus-1 (HSV-1), the egress complex comprises UL31and UL34 (Figure 2). Remarkably, UL31/34 can induce NEbudding on transfection into mammalian cells in the com-plete absence of a viral infection [9]. Less is known aboutthe fission events that produce the vesicular intermediate.The degree to which host cell factors are involved in fissionand other egress reactions remains to be established. Onceformed, HSV-1 glycoprotein B presumably acts as a fusionprotein to execute the fusion of the vesicular intermediatewith the ONM [10] (Figures 1a and 2). However, similarstudies centered on pseudorabies virus and human cyto-megalovirus (HCMV) do not support an essential function

mature virion from the cell.

Ubiquitin proteasome system (UPS): cellular degradation pathway that utilizes

ubiquitin as a tag to signal the degradation of small soluble and membrane-

bound proteins. Proteins marked for degradation by ubiquitin are targeted to

the proteasome, a large protein complex that proteolyzes the target protein.

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Cytoplasm

PM

Secondaryenvelopment

Herpesviruscapsid

Nucleoplasm

Lysosome

Cytosolicaggregates

Autophagosome

Isola�onmembrane

Nuclearaggregates

(b)

(a)

TRENDS in Cell Biology

Figure 1. Egress of herpesvirus capsids from the nucleus: a paradigm for the delivery of nuclear protein aggregates to the cytosolic autophagy machinery? (a) Herpesvirus

capsids are assembled in the nucleus and bud into the perinuclear space to form a membrane-enveloped intermediate, which then fuses with the outer nuclear membrane

(ONM) to release the naked capsid into the cytosol, where the glycoprotein coat is acquired. (b) Using the same route, nuclear aggregates may be transported to the

autophagic machinery that is confined to the cytosol. Note that secondary envelopment, depicted in (a), is topologically equivalent to isolation membrane formation at the

onset of autophagy, depicted in (b). The link to autophagy is at present hypothetical and awaits experimental validation (main text and Box 1).

LINC complex

KASH

?

SunLBR

LmnUS3

UL31

UL34 gB?(iii)

(iv)

(v)

PNS

?

(ii)

PKC(i) Capsid

P

P

TRENDS in Cell Biology

Figure 2. Manipulation of the nuclear envelope during viral egress. Dissolution of the nuclear lamina by local phosphorylation of nuclear lamins via kinases (i) enables the

capsid to gain access to the inner nuclear membrane and to dock onto the nuclear egress complex (ii). Budding (iii) and fission (iv) produce a vesicular intermediate, which

fuses the outer nuclear membrane in a reaction that may involve gB (v). Abbreviations: LINC, Linker of Nucleoskeleton and Cytoskeleton; KASH, Klarsicht, ANC-1, SYNE1

homology; Lmn, lamins; LBR, Lamin B receptor; PKC, protein kinase C; gB, glycoprotein B. Cellular proteins are depicted in blue, viral proteins in red/orange. Note that the

capsid is not drawn to scale; its diameter is approximately three times the width of the perinuclear space (PNS).

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of glycoprotein B in nuclear egress [11,12], suggesting thatother, possibly cellular, factors mediate fusion.

Long believed to be a virus-specific pathway, recentevidence now suggests that nuclear egress might be acellular pathway that has been co-opted by herpesviruses[13]. As we explain below, we believe that this pathwaymay fill a major gap in our understanding of cellularprotein quality control.

A pathway for nuclear protein aggregatesThe proteome of every living organism is surveyed by anelaborate protein quality control system that serves torepair or eliminate misfolded proteins before they reachcytotoxic levels. The major routes for protein turnover inthe cytosol of eukaryotes are the ubiquitin-proteasomesystem (UPS) and autophagy. Although these two systemscooperate on many levels, there is a key difference inselectivity with respect to the physical state of the sub-strate: soluble misfolded species are usually targeted to theUPS for degradation, whereas insoluble protein aggregatesthat cannot be handled by the proteasome are engulfed byan isolation membrane at the onset of autophagy [14].Following the formation of a double membrane structure,the contents of the autophagosome are ultimately deliv-ered to the lysosome via membrane fusion [4] (Figure 1b).

Because the autophagy machinery is confined to thecytosol in interphase cells (in a neuron, for example, no NEbreakdown occurs for decades), it is difficult to envisionhow the cytoplasmic machinery for autophagy can gainaccess to the nucleoplasm, separated from it by the NE.How protein aggregates are handled in the nucleus – thelargest organelle by far in many cell types – is thereforelargely unknown.

We hypothesize that nuclear protein aggregates budinto the intermembrane space of the NE, followed by fusionwith the ONM for cytosolic release. Once inside the cytosol,aggregates are cleared by means of autophagy (Figure 1b).

This postulated mechanism, which we designate egressof nuclear aggregates (EGNA), derives support from thefollowing considerations.(i) The autophagic machinery is confined to the cytosol

and there is no known mechanism that accounts forremoval of protein aggregates in the nucleus.Specifically, a potent nuclear disaggregating activity,which would be required to disassemble nuclearaggregates, has not been described in mammaliancells.

(ii) The NPC does not allow the passage of structuresgreater than 39 nm in diameter [2], suggesting thataggregated protein structures must exit via alterna-tive mechanisms. Nuclear inclusions that exceed thetransport limit have been reported in trinucleotiderepeat disorders [15] and in response to missensemutations [16].

(iii) Perinuclear autophagosomes and upregulation ofautophagy markers are present in cells that expresspathogenic alleles encoding the INM/lamina proteinsEmerin and Lamin A [17].

(iv) Profound thickening of the nuclear lamina is ahallmark of cells from patients with progeriasyndromes [18,19]. This phenomenon may be caused

by aberrant protein deposition – visible as electron-dense material in electron micrographs of those cells –although this remains speculative.

(v) In yeast, nucleus–vacuole (NV) junctions are assem-bled via membrane budding, fission, and fusionevents, allowing the delivery of nuclear componentsto the vacuole in a process termed piecemealmicroautophagy of the nucleus [20,21].

One may therefore entertain the idea that herpes-viruses did not establish this pathway de novo. We hypoth-esize that nuclear egress represents a conserved cellularpathway subverted by herpesviruses to their own advan-tage. Indeed, blebbing-related nuclear transport pathwayshave been considered [13,22,23], but not as part of cellularquality control.

Export of RNA granules resembles nuclear egress:lessons from Drosophila

A recent discovery lends credence to the idea of EGNA. Itwas reported that ribonucleoprotein (RNP) granules in theneuromuscular junctions of Drosophila exit the nucleus ina manner similar to the nuclear egress of HSV-1 capsids[13]. These specific RNP granules experience a size predic-ament similar to that of virus nucleocapsids, in that theyexceed the size limit for exit via nuclear pores. On studyingthe Frizzled nuclear import pathway, the authors discov-ered that D-Frizzled2 (DFz2) foci in the nucleus colocalizedwith RNA and Drosophila A-type Lamin C (LamC). Onelectron microscopy, these foci corresponded to membrane-enclosed granular intermediates within the PNS. Some ofthese granules were present immediately outside the nu-cleus, implying that they can be translocated across themembrane. LamC and an atypical protein kinase C (PKC)isoform are required for the formation of these foci. How-ever, it remains to be established whether RNP export isentirely independent from NPC-dependent transport. Giv-en that PKC is also required in HSV-1 nuclear egress andthat both pathways rely on vesicles as transport inter-mediates in the PNS, one may speculate that HSV-1hijacks a cellular pathway to leave the nucleus [13].

We argue that aggregated nuclear proteins would like-wise be too large to exit the nucleus via nuclear pores.Instead, utilization of a budding pathway through the NEwould allow access to the cytoplasm and the known mech-anisms of autophagy. Given that cytosolic protein aggre-gates are actively transported to a perinuclear site [24,25],the transport routes of cytosolic and nuclear protein aggre-gates might converge at such a site, followed by theirsequestration into autophagosomes (Figure 1b).

Cellular factors implicated in nuclear egressA mechanistic understanding of this type of nuclear egressis in its infancy and many crucial players await identifica-tion. The use of viruses as tools has illuminated manycellular transport pathways [26]. Herpesviruses maytherefore prove helpful in dissecting both cellular and viralaspects of nuclear egress. By analogy to viral manipulationof the NE, the cellular egress pathway may comprise thefollowing steps (Figure 2): (i) local dissolution of nuclearlamina; (ii) recognition of cargo by a receptor at the INM;

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(iii) budding into the PNS; (iv) fission to yield a vesicularintermediate; and (v) fusion with the ONM.

Although cargo receptors remain elusive, the viral nu-clear egress complex interacts with the Lamin B receptorindirectly leading to its relocalization [27,28]. The func-tional implications of this observation remain to be estab-lished. More is known about the action of kinases, whichfunction in a manner analogous to NE breakdown duringmitosis [29]; PKC-type kinases are involved in lamin phos-phorylation to facilitate egress [6,13]. The mechanism thatensures that those kinases only act locally to prevent bulkvesiculation of the NE are unknown, but is likely to involverecruitment to egress sites defined by specific, INM-resi-dent receptors.

Which cellular components aid in membrane curvature,fission, and fusion, and how is the transport process ener-gized? Topologically, the budding reaction is similar to theformation of a multivesicular body (MVB) in endosomes,responsible for the formation of intraluminal vesicles[30,31]. CHMP1, a member of the Snf7 family requiredfor MVB formation, was first identified as a nuclear protein[32]. Manipulation of the MVB machinery compromisesHSV-1 production [33,34], but whether MVB componentsaffect only secondary envelopment in the cytosol or serveanother function during nuclear egress is not known.

Many AAA+ (ATPases associated with a variety ofcellular activities) ATPases [35] play important roles inmembrane remodeling; examples include VPS4, a criticalcomponent of the MVB machinery [30,31] and NSF, whichdisassembles SNARE proteins after membrane fusion [36].There are several reasons why Torsin AAA+ ATPases arelikely to be functionally important (four are encoded in thehuman genome: TorsinA, TorsinB, Torsin2A, and Tor-sin3A). First, to our knowledge they are the only AAA+ATPases found in the PNS. Second, vesicular structures inthe PNS resembling viral egress intermediates are visiblein electron micrographs derived from mouse models ofDYT1 dystonia, a disorder caused by a mutation in theTOR1A gene, as well as in primary cells of primary dysto-nia patients [37–41]. TorsinA – the product of the TOR1Agene – and possibly other Torsins may therefore play amajor role in the formation or resolution of these vesicularstructures [13,42]. Consistent with this idea, expression ofdominant negative alleles of Torsin A and B potentlyreduce HSV-1 production [43].

Apart from the nuclear lamina, another obstacle tonuclear egress that must be modulated is the Linker ofNucleoskeleton and Cytoskeleton (LINC) complex. Thiscomplex serves as a molecular ruler to restrict the widthof the PNS to approximately 40 nm. It comprises a trimericSUN (Sad1/UNC84) protein that spans the INM and bindsthe lamin network with its N terminus, while bindingNesprin/KASH (Klarsicht, ANC-1, SYNE1 homology) pro-teins in the PNS [44–46]. These Nesprin proteins, whichare the second component, span the ONM and attach to thecytoskeleton with their N termini [45,47]. The precisenumber and density of LINC complexes in the nuclearmembrane is unknown, but it is likely that the LINCcomplex would need to be modulated (i.e., degraded, dis-sociated, or relocalized) to permit the formation of vesicu-lar intermediates, the size of which exceed the typical

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width of the PNS by a factor of approximately three.Indeed, Sun2 is degraded during HCMV infection [48],indicating that herpesviruses are capable of manipulatingthese complexes. The LINC complex might otherwise playan active role in the nuclear egress pathway; for example,by connecting exported cargo to cytoskeletal motor pro-teins for subsequent transport to their final destination.

The fact that TorsinA physically interacts with nesprin-3 and affects the localization of Sun2 suggests that Torsinsmay manipulate the LINC complex [49,50]. The precisefunction of Torsins remains to be defined, but is likely toinvolve at least one of the following: disassembly of theLINC complex; disassembly/recycling of fusion proteinsanalogous to NSF [36]; a role in membrane fission; orimposition of membrane curvature. Ultimately, in vitroreconstitution of the egress reaction will be critical fordistinguishing among these possibilities. A prerequisiteto this approach is that the identities of most componentsmust be known, which is a major limitation at this time.That many of these components are likely to be membraneproteins, which are often difficult to purify in their nativestate, will probably be another complication. Of note, MVBformation, a similarly complicated and topologically relat-ed process, was successfully reconstituted in vitro [51],suggesting that biochemical reconstitution lies withinthe realm of possibilities.

Implication for laminopathies and related diseasesaffecting NE morphologyThere are many examples of diseases caused by mutationsin INM proteins or their interacting partners, collectivelydesignated nuclear envelopathies or laminopathies[52,53]. Among the most severe manifestations are proge-ria (premature aging) syndromes and muscular dystro-phies. Autosomal dominant (AD)-Emery–Dreifussmuscular dystrophy (EDMD) is caused by distinct LaminA mutations, whereas X-linked EDMD (X-EDMD) iscaused by mutations in the emerin (EMD) gene, an integralprotein of the NE. AD-EDMD is more common than X-EDMD and is often more severe, with earlier onset, often invery early childhood. Both forms are similar in that theypresent with muscle weakness and wasting and earlycontractures of the elbow, the Achilles tendons, and thespine. Eventually, cardiac conduction defects can lead tocomplete heart block and death in EDMD patients [54].

Previous studies of laminopathies focused on altera-tions in gene expression, mechanical stability of the nucle-us, or cell cycle regulation. However, the underlyingmolecular mechanisms that give rise to pathology remainpoorly understood [55]. Several pathogenic alleles thatcause laminopathies act as dominant negatives to yieldprotein aggregates when overexpressed, and aberrantintranuclear foci have been observed in primary cells frompatients [53,55,56]. Lamin A-deficient mouse models donot show obvious phenotypes [57], suggesting a gain-of-function mechanism, possibly through proteotoxicity. Pro-teinaceous deposits are a hallmark of many neurodegen-erative diseases induced by proteotoxic – and oftendominant – variants that create structures resistant tothe cellular machinery responsible for repair and turnoverof damaged proteins [58,59]. There may be a link between

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these diseases and the LINC complex. Mice lackingnesprin-1, a KASH protein in the ONM, display anEDMD-like phenotype [60]. Mutants causing both formsof EDMD and progeria bind Sun1 and/or Sun2 less tightly[61,62]. Sun1 is upregulated in mouse models of AD-EDMDand in primary cells of progeria patients, and geneticablation of Sun1 from the animals reduces the toxicity ofthe disease alleles [63].

Thus, the aforementioned diseases deserve scrutinyfrom the perspective of nuclear egress. Recent seminalfindings [13] provide compelling evidence for interferenceof RNP granule export caused by an aberrant Lamin Callele and thus establish the idea that the toxicity exertedby laminopathy-associated alleles may be attributed atleast in part to interference with the nuclear egress path-way [13]. Whether this also applies to the turnover ofnuclear protein aggregates remains to be established. Itis possible that the wide variety of symptoms manifested indifferent laminopathies is due to differential effects of themutations in question on the egress of various types ofcargo from the nucleus.

Concluding remarksBy analogy to nuclear transport via the NPC, nuclearegress may be a versatile transport mechanism with theadded advantage of an increased cargo size limit. Aninterdisciplinary effort will be required to identify theunderlying machinery and to determine whether viraland cellular pathways indeed overlap substantially withrespect to the mechanistic underpinnings and shared com-ponents. It will also be interesting to explore the additionalbiological functions of this unusual transport route. Acomplementary approach to classical genetics could entailscrutinizing the interactions of viral proteins with as yetunknown host cell proteins (Box 1). Determining the func-tional consequences of manipulating these interactions, forexample by RNA interference, will aid in the identificationand validation of key players in the nuclear egress ma-chinery (Box 1). Suitable nuclear model substrates that canbe tracked biochemically and via live cell imaging will haveto be established to validate the existence of nuclear

Box 1. Outstanding questions

� Is it possible to exploit herpesviruses to find the cellular

components of the endogenous nuclear egress pathway and

elucidate the molecular mechanism of transport?

� Which proteins are responsible for generating the membrane

curvature, fission, and fusion required for the nuclear egress

pathway? Is ESCRTIII involved in creating the negative curvature

required to vesiculate the INM?

� What happens to the LINC complex when the nuclear egress

pathway is activated? Is there a factor responsible for its

disassembly, degradation, or relocalization?

� Which functions are fulfilled by Torsin ATPases in the context of

nuclear egress?

� Are there other substrates that need to exit the nucleus but are too

large for transport through the NPC? Photoconvertible model

substrates suitable for live cell imaging will be required to provide

conclusive evidence for nuclear egress.

� Is transport unidirectional or can large components located in the

cytoplasm enter the nucleus via the same pathway?

aggregate egress (Box 1). The breakdown of these sub-strates should be insensitive to nuclear export inhibitors,but sensitive to inhibitors of the autophagy pathway.

Future work will reveal whether these criteria arematched, which would strongly support the idea of a func-tional link between the nucleus and autophagy machinery.The basic components of the egress machinery may consti-tute novel drug targets and present therapeutic opportu-nities for treatment of viral infections and laminopathies.

AcknowledgmentsWe thank Mark Hochstrasser and members of the Schlieker laboratoryfor critical reading of the manuscript and Tom DiCesare for help withillustrations. C.S. is supported by the Ellison Medical Foundation (AG-NS-0662-10) and the National Institutes of Health (DP2 OD008624-01).

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