highway to the inner nuclear membrane: rules for the road

There has been a recent resurgence of interest surrounding the diverse functions of integral membrane proteins at the nuclear envelope (NE), with an emphasis on those proteins that are localized at the inner nuclear membrane (INM). One central reason for this renewed focus is that INM proteins, particularly those associated with lamins, are linked to a multitude of genetic diseases1,2. Evaluating the targeting of INM proteins will therefore be an important aspect of understanding the mechanisms of these diseases. To reach the INM, membrane proteins move across the three continuous membrane domains that make up the NE: the outer nuclear membrane (ONM), the pore membrane (POM) and, last, the INM (where they then reside) (BOX 1). The ONM is a continuation of the ribosome-studded rough endoplasmic reticulum (ER), but also contains ONM-specific protein complexes3,4. The INM faces the nucleoplasm and hosts a number of specific proteins that interface directly with the genome2,5. The INM is con-nected to the ONM via the POM, where large macromolecular assemblies called nuclear pore complexes (NPCs), which control the passage of molecules to and from the nucleus6, are inserted (FIG. 1).

NPCs are anchored in the POM by transmembrane proteins that extend into the lumen of the NE. Cytosolic, transmembrane and lumenal domains of membrane proteins must all navigate past the substantial scaffold of the NPC to reach the INM. Therefore, the

NPC might regulate access of integral mem-brane proteins to the INM. Recent evidence in both yeast and higher eukaryotes indicates that, in addition to diffusion, active signal-mediated targeting can contribute to the passage of integral INM proteins across the POM. Here we integrate what is known about the path across the POM and propose a set of guidelines for access to the INM. Surprisingly, these guidelines resemble those that control the nuclear import of soluble proteins.

Access to the INM

Integral INM proteins were first identified in metazoans in which the nuclear lamina, a meshwork of intermediate filament lamin proteins, lines the surface of the INM2,5. Early experiments that examined the target-ing of integral INM proteins were performed by Powell and Burke7, and these laid the groundwork for a central theme in the field: INM proteins are retained once they reach the INM by interactions with either the lamina or chrom atin. They showed that lamina-associated polypeptide-1 (LAP1) required the expression of the lamin A gene in order to be targeted to the NE, which

indicated that the lamin A protein conferred the retention of LAP1 at the INM. This model is further supported by observed physical interactions between INM proteins and the nuclear lamina, and by changes in mobility that are observed when INM proteins reach the INM2,5,8–13. In addition, the steady-state localization of the INM protein emerin and perhaps the INM SUN proteins are altered in lamin-knock-down or lamin-null cell lines13–18. Therefore, the lamina serves as an important platform for the organization of the INM in metazoa. In principle, nuclear reten-tion can also be mediated by interactions with other nuclear proteins or with chromatin, which is perhaps most relevant in organisms that lack lamins, such as yeasts.

The POM as a diffusion barrier. For nuclear retention to be sufficient to explain the mechanism of targeting integral INM proteins, movement across the POM must occur by diffusion. Viral membrane proteins with small extralumenal domains can access the INM, but these do not accumulate there, indicating that free diffusion can occur across the POM19–21. One predic-tion of a diffusion-based model is that the likelihood that a given protein will diffuse across the POM will depend on the size of its hydrophilic domains. As hydrophilic domains increase in size, they are more likely to encounter proteins of the NPC (termed nucleoporins, FIG.1) that might impose a barrier to their diffusion. This might explain why artificially increasing the size of nucleoplasmic domains of INM proteins to >60–75 kDa prevents their access to the INM9,11,22. There is, however, little evidence that membrane proteins with large extralumenal domains can access the INM by diffusion alone. A landmark paper by Ohba et al.9 showed that the targeting of an INM protein reporter in HeLa cells is energy dependent. By either chilling cells or using energy poisons, the accumulation of the reporter in the INM was inhibited. These data provided the first compelling evidence that diffusion was not the only means of passage across the POM — a finding that re-invigorated the field and opened the door for further investigations into the mechanisms that regulate this active pathway.

O P I N I O N

Highway to the inner nuclear membrane: rules for the roadC. Patrick Lusk, Günter Blobel and Megan C. King

Abstract | To enter the nucleus a protein must be chaperoned by a transport factor

through the nuclear pore complex or it must be small enough to pass through by

diffusion. Although these principles have long described the nuclear import

of soluble proteins, recent evidence indicates that they also apply to the import of

integral inner nuclear membrane proteins. Here we develop a set of rules that

might govern the transport of proteins to the inner nuclear membrane.

...we develop a set of rules that might govern the transport of proteins to the inner nuclear membrane.

414 | MAY 2007 | VOLUME 8 www.nature.com/reviews/molcellbio

PERSPECTIVES

© 2007 Nature Publishing Group

POMONM

INM

Doa10

Doa10

Mps2/Mps3Nem1/Spo7

Prm3Emerin LaminaNesprins

Nesprins

Heh1/Heh2 MAN1LAP2βLAP1Asi1–Asi3

NurimSUN1LBR

Nem1/Spo7

SPB

NucleusYeast Mammalian

Cytoplasm

ER ER

Active INM protein targeting

A number of potential mechanisms might govern an active pathway for integral mem-brane proteins to reach the INM. In one model, ATP-driven changes in nucleoporin interactions might allow membrane proteins to travel across the POM9. In an alternative model, integral INM proteins might directly, or through an associated factor, interact with specific nucleoporins to promote their movement past the NPC. An important component of the latter model might be the specific recognition of discrete signal sequences (perhaps nuclear-localization signals (NLSs)) on cargo molecules by a dedicated transport machinery. Soullam and Worman22 sought to define such a signal by studying the targeting of chicken

lamin B receptor (LBR). They found that the N-terminal nucleoplasmic domain of LBR contained the sequence information required for INM targeting, and LBR could also be efficiently targeted to the nucleus as a truncated, soluble protein. Further, they recognized that the targeting domain of LBR contains a sequence that resembles a classi-cal (c)NLS (see below). However, a chimeric protein consisting of a cNLS fused to a transmembrane domain was insufficient to confer INM targeting, leaving unanswered whether the cNLS was required for efficient targeting of LBR. Despite this, a potential role for cNLS-like sequences in INM targeting is difficult to discount, as putative NLSs can be predicted in the majority of known integral INM proteins in both yeast

and higher eukaryotes (TABLE 1). Further, soluble nucleoplasmic domains of other integral INM proteins (of various sizes) also accumulate in the nucleus, which suggests the presence of NLSs in these regions23,24. Taken together, these observations suggest a potential role for the soluble-protein import machinery in the targeting of NLS-bearing integral INM proteins.

Soluble-protein import. The proposal that integral INM proteins can share the nuclear transport machinery with soluble proteins leads us to consider the mechanism of soluble-protein import. In a simplified model, the active nuclear import of soluble molecules proceeds in three steps: cargo recognition, translocation through the NPC and cargo release (FIG. 2). In the first step, a protein bearing an NLS is recognized by a soluble transport factor (karyopherin; also known as importin). NLSs are generally short (<50 amino-acid residues) basic sequences, and an individual NLS can confer an interaction that is specific for one or more karyopherin25. Karyopherins fall into two major families: karyopherin-α and karyopherin-β families. In yeast there are 14 karyopherin-βs and one karyopherin-α (Kapα) These families are larger in metazoa, in which the karyopherin-α homologues in particular have diversified. Both protein families can directly recognize NLSs; in the classical import pathway, however, karyopherin-β1 (Kapβ1) interacts with specific NLSs (cNLSs) indirectly through Kapα (FIG. 2a). This additional layer of complexity could facilitate further levels of regulation for cNLS-mediated import that are not present in pathways in which cargo is directly recognized by members of the karyopherin-β family (FIG. 2b).

In the second step of soluble-protein import, the karyopherin–cargo complex moves through the NPC by interacting with a subset of nucleoporins that are enriched in Phe-Gly (FG) amino-acid residues in repetitive motifs. Members of the karyopherin-β family directly interact with these FG-repeats to promote the translocation of the karyopherin–cargo complex by a mechanism that remains poorly understood25. Although it is thought that the FG-repeats provide an indiscriminate surface for karyopherin-β binding, there are indications that certain nucleoporins can function to regulate specific karyopherin-import pathways26.

Last, once inside the nucleus, the release of cargo is stimulated by the binding of the small GTPase Ran in its GTP-bound form

Box 1 | The yeast and mammalian nuclear envelope

The nuclear envelope (NE) is an extension of the endoplasmic reticulum (ER) and is formed by three connected membrane domains: the outer nuclear membrane (ONM), the pore membrane (POM) and the inner nuclear membrane (INM). The nuclear pore complex (NPC, centre) is inserted into the POM, where it regulates nucleocytoplasmic transport. Integral membrane proteins that reside and function at the INM in both yeast and mammals are shown.

Yeast INM proteins. A related family of RING-domain-containing membrane proteins (Asi1, Asi2 and Asi3) function as regulators of amino-acid-uptake signalling44,45. Doa10, which also contains a RING domain, targets nuclear proteins for degradation, but localizes to both the INM and the ONM/ER41. Nuclear envelope morphology-1 (Nem1) and Spo7 form a phosphatase complex that regulates lipid biosynthesis46. Mps2 (REF. 47) and Mps3 (REF. 48) are shared components of the INM and spindle pole body (SPB). Deletion of Prm3 (REF. 24) causes defects in the fusion of nuclei during sexual reproduction. Helix–extension–helix-1 (Heh1) and Heh2 are homologues of mammalian MAN1 (REF. 2,5), but their role in yeast is not yet understood29.

Mammalian INM proteins. In mammals, the INM is lined with a polymer of the intermediate filament proteins called lamins. The lamina-associated polypeptides LAP1 and LAP2β and lamin B receptor (LBR) constitute the major lamin-binding proteins at the INM. LAP2β, emerin and MAN1 contain the conserved LEM domain, which interacts with the DNA-binding protein barrier-to-autointegration factor (BAF). Defects in emerin lead to Emery–Dreifuss muscular dystrophy. MAN1 antagonizes SMAD signalling. SUN1 and SUN2 (which each contain the SUN domain) are homologous to the yeast protein Mps3 and serve as tethers for the KASH-domain (Klarsicht, Anc-1 and Syne homology) proteins (including nesprins) that reside specifically at the ONM. KASH-domain proteins link the NE to the cytoskeleton. Additional INM proteins have been identified (such as nurim), but remain to be functionally characterized. A number of proteomics approaches have identified additional putative INM proteins49,50. For reviews of metazoan INM proteins, see REFS 2,5.

P E R S P E C T I V E S

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | MAY 2007 | 415


Nup170Nup188

NUP155NUP205

Pom152, Pom34,Ndc1

Pom152, Pom34,Ndc1

gp210, POM121,NDC1

gp210, POM121,NDC1

NUP50Nup2

Nup170Nup188

NUP155NUP205

FG FG

FGFG

FG FG

FG

FG

FG

FG

FG

FG

Centralchannel

Nuclearbasket

Cytoplasmicfilaments

Cytoplasm

NucleusYeast Mammalian

INM

ONM/ERPOM

to karyopherin-β (FIG. 2). Cargo release in the Kapα–Kapβ1-mediated import pathway is also stimulated by binding to a specific nucleoporin on the nuclear face of the NPC27,28 (nucleoporin-2 (Nup2) in yeast, NUP50 in mammals) (FIG. 2a).

NLSs in integral INM protein targeting. To test the hypothesis that the soluble transport machinery might be involved in targeting membrane proteins to the INM, we exam-ined the role of NLSs in yeast INM proteins. We focused on the protein helix–extension–helix-2 (Heh2, named after a domain found at the N terminus of proteins in this family) because of its homology to well characterized mammalian INM proteins MAN1 and LEM2 (BOX 1). Either mutating or deleting the NLS of Heh2 (or of Ydl089w, an INM protein of unknown function) caused the dispersal of the mutated proteins throughout the ER29. Most strikingly, as shown by immuno-electron microscopy, Heh2 that lacks the NLS was virtually excluded from the INM. These

data provide strong support for the idea that an NLS in these proteins is essential for them to travel across the POM.

Kapα–Kapβ1 in integral INM protein targeting. The NLS examined in Heh2 (and the type of NLS predicted in many INM cargoes, TABLE 1) conforms to the canonical cNLS, which binds Kapβ1 indirectly through Kapα. Consistent with this, we observed that yeast strains that harbour mutations in either Kapα or Kapβ1, but not in several other karyopherin-β mutants, disrupt targeting of Heh2 and Heh1 (a paralogue of Heh2), which indicates that these cargoes are transported specifically by the Kapα–Kapβ1 pathway29. Further evidence of specificity for the Kapα–Kapβ1 pathway can be drawn from experiments in which the NLS of Heh2 was replaced by NLSs that are recognized directly by other karyopherin-β family members. These NLSs failed to target Heh2 to the INM, whereas Kapα-specific NLSs mediated this targeting. The interchange-

ability of NLSs is a common characteristic of soluble transport; specificity for Kapα NLSs in integral membrane protein transport might reflect unique aspects of this pathway across the POM.

Nucleoporins at the POM

Understanding how integral INM proteins reach the INM hinges on examining the specific interactions of INM proteins with nucleoporins as they migrate across the POM, whether via a passive or active mechanism.FG-nucleoporins in INM protein import. As discussed above, the interactions between karyopherin-βs and FG-containing nucleo-porins are necessary to import soluble pro-teins. Therefore, do FG-nucleoporins also contribute to karyopherin-mediated import of membrane proteins?

In a model in which FG-domains are involved in the import of integral membrane proteins to the INM, the karyo-pherin–INM cargo complex will interact with FG-nucleoporins localized near the POM, or with FG-nucleoporins that are able to adopt a conformation in which the FG-repeats could reach the POM region. The majority of the FG-repeat containing nucleoporins are concentrated on the cyto-plasmic and nuclear faces of the NPC30 and are also thought to line the central channel (FIG. 1). In general, FG-repeat nucleoporins are not components of the nucleoporin sub-complexes that reside nearest to the POM6 (the one exception might be the vertebrate membrane FG-nucleoporin POM121 (POM protein of 121 kDa)). Therefore, whether FG-proteins can interact with integral mem-brane proteins as they move across the POM is unclear. FG-domains, however, are natively unfolded31, highly flexible32 and can sample a large conformational space that might extend to the NE. The likelihood of a particular FG-domain extending to the NE probably varies for each individual FG-nucleoporin. So, different FG-nucleoporins might prefer-entially contribute to the transport of soluble proteins through the central channel, and to the transport of INM proteins across the POM. This might explain the observation that wheat germ agglutinin, which binds to a subset of FG-nucleoporins modified by O-linked N-acetylglucosamine mono-saccharides, inhibits soluble transport but fails to disrupt targeting of an INM protein reporter9.

Understanding the role of specific FG-nucleoporins in the transport of membrane proteins to the INM represents a significant challenge, as there is remarkable

Figure 1 | Architecture of the nuclear pore complex. Architecture of yeast (left) and mammalian

(right) nuclear pore complexes (NPCs). NPCs mediate the bidirectional exchange of molecules

between the cytoplasm and the nucleus. In addition, membrane proteins destined for the inner nuclear

membrane (INM) must move from the outer nuclear membrane (ONM) across the pore membrane

(POM). The general architecture of the NPC consists of nucleoporins that contain repetitive motifs of

Phe-Gly (FG) amino-acid residues (green) that sit on a scaffold of non-FG-nucleoporins (including

Nup170 (nucleoporin of 170 kDa) and Nup188 (their mammalian homologues, NUP155 and NUP205,

are shown on the right). The scaffold is embedded into the POM by integral POM nucleoporins

(Pom152 (POM protein of 152 kDa), Pom34, Ndc1 (nuclear division cycle-1) in yeast and gp210 (glyco-

protein of 210 kDa), POM121 and NDC1 in mammals). The FG-nucleoporins are thought to create a

continuous surface of FG-repeats that extend from the cytoplasmic filaments through the central

channel to the nuclear basket. Nup170, Nup2, Nup188, Pom152 and gp210 have been shown to func-

tion in INM protein transport. ER, endoplasmic reticulum.




Table 1 | Prediction of nuclear-localization signals in inner nuclear membrane proteins

Name* TM segments Extralumenal domain size (aa)

PSORT II‡ NLS predictions Lumenal domain size (aa)

Refs

Human INM proteins

MAN1 2 471 (NT) 190-RRKP; 285-RPRR 233 2,5

262 (CT) 706-PHDRKKM

LAP2β 1 410 (NT) 258-PRKRVET 23 2,5

LAP1A 1 337 (NT) 80-PVGKRTR; 111-RRQPRPQETEEMKTRRT

22 2,5

LAP2γ 1 301 (NT) None predicted 23 2,5

TMEM43/LUMA§ 4 258 (CT) 111-PAVKLRR <20 2,5

Emerin 1 224 (NT) 31-RRLYEKKIFEYETQRRR 7 2,5

LBR 8 208 (NT) 63-RKGGSTSSPSRRRGSR; 72-PSRRRGS: 95-RRSASASHQADIKEARR; 169-RPRR

<50 2,5

LEM2 2 208 (NT) 126-PAQLRRR 141 51

208 (CT) 450-PPQSRRM

SUN1 3 176 (NT) None predicted 577 2,5

SUN2 3 158 (NT) 102-RRRR 482 2,5

RFBP§ 8 or 9 <65 None predicted ≤ 504 2,5

Nurim 6 <40 None predicted <40 2,5

Nesprin-1 and -2¶ See below¶ See below¶ 2,5

Saccharomyces cerevisiae INM proteins

Heh1 2 453 (NT) 86-PRRSRRA; 93-RREKSASPMAKQFKKNR; 173-RKKRK; 216-KKRK

29

110 (CT) 662-PRQKRHL

Asi3 5 77 (NT) None predicted 44

382 (CT) 427-KKPRVGKRKKR; 434-RKKRDLNKYVTEKNYKK

Asi1 5 329 (CT) None predicted <75 45

Nem1 2 86 (NT) 78-PKKPKAL <10 46

319 (CT) 238-KKLIPKSVLNTQKKKKL

Heh2 2 316 (NT) 102-KRKR; 124-PKKKRKKR

206 29

97 (CT) None predicted

Mps2 1 308 (NT) 142-PRKK; 282-KRKH 67 47

Doa10 14 <251 None predicted <51 52

Asi2 2 230 (NT); 31 (CT) None predicted <10 44

Ydl089w 4 53 (NT); 89 (TM 2–3) None predicted 70 29

205 (CT) 475-PKKKK

Mps3 1 153 (NT) None predicted 507 47,48

Pga1§ 2 131 (TM 1–2) 37-KKPR <20

Spo7 2 71 (NT) 24-PRRR 8 46

127 (CT) 246-RRRK

Prm3 1 108 (NT) 68-RKHKTTTSSTKSRTKSK 7 24

Gtt3§ 2 60 (TM 1–2) None predicted 240

*Inner nuclear membrane (INM) proteins are listed in order of decreasing extralumenal domain size. Nucleoplasmic domains are listed as NT (N-terminal), CT (C-terminal) or as

between transmembrane (TM) segments (for example, TM1–2 is between transmembrane segment 1 and 2). The number of TM segments in each membrane protein is listed. ‡Nuclear-localization signals (NLSs) in the listed proteins were predicted using the PSORT II algorithm. NLS positions are given as the number of the first amino acid of the

putative NLS. §Predicted integral INM proteins. ¶Nesprin-1 and -2 have many isoforms, some of which have NLSs. It is not clear which isoforms have access to the INM53,54.

aa, amino acids; Gtt, glutathione transferase; Heh, helix–extension–helix; LAP, lamina-associated polypeptide; LBR, lamin B receptor; RFBP, RING-finger binding protein.




Nup2/NUP50

a Import of cargo recognized by a karyopherin-α

b Import of cargo recognized by a karyopherin-β

Nucleus

Cytoplasm

ONM

INM

ONM

INM

cNLS

RanGTPNLS Kapβ1Karyopherin-βKaryopherin-α Transmembraneprotein

functional redundancy between the FG-regions of nucleoporins33. Nonetheless, although deletions of subsets of FG-regions have little effect on Kapα–Kapβ1-dependent import33, they do perturb other karyopherin-β import pathways. So, the Kapα–Kapβ1 complex might be able to interact with FG-regions that other karyopherin-βs cannot interact with, and this might explain the observed specificity to the Kapα–Kapβ1 pathway observed for the targeting of integral INM proteins29.

Non-FG-nucleoporins in INM protein import. In addition to the potential role of FG-nucleoporins in the transport of membrane proteins across the POM, we must consider that the POM is occupied predominantly by non-FG-nucleoporins6 (FIG. 1). Do these nucleoporins also facilitate the passage of karyopherin–INM-cargo complexes across the POM?

In higher eukaryotes, antibodies directed against the integral membrane nucleoporin gp210 (glycoprotein of

210 kDa; also known as NUP210) can dis-rupt the targeting of INM cargo9. In yeast, deletion of NUP170, a non-FG-nucleoporin that functionally interacts with integral membrane proteins of the POM34–36 and is conserved throughout all eukaryotes, results in a significant delocalization of at least Heh1 and Heh2 from the INM29. Importantly, disruption of NUP170 does not dramatically affect Kapα–Kapβ1-mediated soluble nuclear transport, although it does increase the molecular mass limit for diffusion of soluble pro-teins37. We propose that Nup170 might direct specific rearrangements of the NPC that allow for the passage of INM proteins: this is similar to the interaction of Nup170 with the neighbouring nucleoporin, Nup53, which is modulated as cells progress through the cell cycle38.

In considering the role of non-FG-nucleoporins, we can also explore the possibility that Kapα promotes the passage of INM cargo through direct interactions with nucleoporins independently of Kapβ1.

Although a novel concept, it provides another explanation for the specificity to the Kapα pathway observed by King et al. and is further supported by the observa-tion that viral membrane proteins that are targeted to the INM are associated with a truncated Kapα-like homologue in Sf9 insect cells39. This Kapα-like protein (importin-α16) is much smaller (15 kDa) than other karyopherin-αs (60 kDa) and lacks the region responsible for binding to Kapβ1. There is also evidence for a Kapβ1-independent pathway for trafficking of Kapα into the nucleus, but the mechanistic basis for this transport is unknown40.

Passive diffusion across the POM. Although we have focused on the role of nucleo porins in facilitating karyopherin-mediated import of integral INM proteins, disruption of nucleo porin function is also likely to compromise NPC structure such that the diffusion of integral membrane proteins across the POM is obstructed. Consistent with this, Nup188 and Pom152 (both non-FG-containing nucleoporins) appear to affect passive access of membrane proteins to the INM29,41.

The rules for the road

We propose that the pathway for passage across the POM in both yeast and higher eukaryotes is bimodal: it uses both passive and active mechanisms. On the basis of current evidence20,29,41, we estimate that proteins with nucleoplasmic domains that are <~25 kDa (the secondary structure will also be important) are able to diffuse across the POM and be retained in the INM by interacting with the lamina or other elements of the nuclear architecture (BOX 2a,b). A prediction of this model is that integral ER proteins with cytoplasmic domains that are <~25 kDa could localize (and function) in the INM. One example of this is the ubiquitin-ligase Doa10 (TABLE 1). Doa10 was thought to be exclusively an integral ER protein, but can also access the INM, where it drives the degradation of specific intranuclear targets41. Interestingly, artificially increasing the size of an extra-lumenal domain of Doa10 prevents its passage to the INM41, suggesting that its size dictates its exclusion (BOX 2d). Therefore, our understanding of the membrane-protein composition of the INM cannot be limited to proteins that localize exclusively to the INM at steady state; it can be expanded to include proteins that are able to access the INM but that are also distributed throughout the ONM and ER.

Figure 2 | Karyopherin-mediated import. a | Import of cargo that is recognized by a karyopherin-α.

Karyopherin-α directly recognizes both soluble or transmembrane proteins in the outer nuclear mem-

brane (ONM) that bear a classical nuclear-localization signal (cNLS) and is subsequently bound in the

cytoplasm by Kapβ1. This trimeric complex docks and translocates through the nuclear pore complex

(NPC; colouration as in FIG. 1) by a mechanism that is poorly defined. Upon reaching the nucleus, the

dissociation of the cNLS cargo is driven by the concerted action of the binding of the small GTPase

Ran in its GTP-bound form (RanGTP) to Kapβ1, and of Nup2 (NUP50 in mammals) to karyopherin-α.

Ran functions to release the cNLS-bearing cargo from the karyopherin. So far, the active transport of

integral inner nuclear membrane (INM) proteins appears to use this pathway to reach the INM.

b | Import of cargo that is recognized by a karyopherin-β. Non-classical NLSs are recognized directly

by one or more karyopherin-β without an adaptor karyopherin-α. The heterodimer moves through the

NPC, again by a mechanism that is poorly defined, and the NLS-bearing cargo is released primarily by

the binding of RanGTP.




Nucleus

Cytoplasm

LaminaChromatin

LaminaChromatin

ONM/ER

INM

NLS

NLS

NLS

a

b

c

d

e

f

We envision that proteins with soluble domains that are >~25 kDa can only effi-ciently travel across the POM concomitant with changes in nucleoporin–nucleoporin interactions, as first proposed by Ohba et al.9 (BOX 2; compare d and e). Whether karyo-pherins participate in driving these changes, or whether they serve to increase the efficiency with which INM cargoes interact and pass through the NPC, is not yet clear.

In either case, there will also be an upper limit to the size of cargoes that can move across the POM using the active pathway; on the basis of our knowledge of proteins that reside in the INM, this limit is probably ~75 kDa. It is also possible that membrane proteins with nucleoplasmic domains that are >75 kDa can be localized to the INM by associating with nuclear factors before post-mitotic NE assembly is complete in metazoa.

Perhaps more so than for soluble transport, the position of the NLS in a membrane protein might be important for INM targeting (given that the NLS will be membrane tethered), and this might explain the inability of an NLS fused to a chimeric membrane protein to be targeted exclusively to the INM22. Alternatively, an NLS might be required for cargo to move across the POM, and another factor (for example, nuclear retention) might be needed for exclusive INM localization at steady state (BOX 2e). Whether an NLS is sufficient to target a membrane protein to the INM might also depend on the affinity of the NLS for Kapα42 (BOX 2f), or an NLS might potentiate INM targeting of proteins already able to cross the POM (BOX 2c). Although we can predict NLSs in the majority of integral INM proteins (TABLE 1), we must also consider the possibil-ity that, like the transport of certain soluble cargoes43, membrane proteins might piggy-back across the POM by interacting with other signal-bearing partners. Furthermore, although we emphasize the importance of Kapα–Kapβ1 in the active pathway across the POM, we cannot exclude the possibil-ity that other karyopherin-βs might also contribute to the targeting of integral INM proteins, as NLSs that are recognized by karyopherin-βs are difficult to predict.

Perspective

We have attempted to integrate studies from higher eukaryotes and yeast to develop a model that describes the composition of the INM across eukaryotes. The most obvious differences between the yeast and metazoan nuclear architectures are the presence of the lamins in metazoa and the size of NPCs. Although the lamina plays a part in the retention of a number of INM proteins, the mechanistic principle of retention can equally apply to any element of the nuclear architec-ture. Furthermore, it is established that there is remarkable conservation between yeast and vertebrates in terms of NPC structure, com-position and, most importantly, the mecha-nisms of transport26. We feel it is unlikely that the targeting of integral INM proteins will be an exception. Understanding how the NPC modulates the POM to accommodate karyopherin–cargo complexes represents a key challenge.

C. Patrick Lusk, Günter Blobel and Megan C. King are at the Laboratory of Cell Biology, Howard Hughes

Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.

Correspondence to M.C.K. e-mail: [email protected]

doi:10.1038/nrm2165

Published online 18 April 2007

Box 2 | Rules for steady-state distribution of nuclear envelope membrane proteins

• Rule a — extralumenal domain(s) <~25 kDa . Membrane proteins with extralumenal domains that are <~25 kD can diffuse throughout all endoplasmic reticulum (ER) subdomains, and therefore localize to both the inner nuclear membrane (INM) and the outer nuclear membrane (ONM)/ER.

• Rule b — extralumenal domain(s) <~25 kDa, INM retention. As the size of their extralumenal domains are <~25 kDa, these proteins can access the INM by diffusion. Once at the INM, they are retained by interactions with the nuclear architecture, resulting in an almost exclusive INM localization.

• Rule c — extralumenal domain(s) <~25 kDa, with a nuclear-localization signal (NLS). Although these membrane proteins can access the INM by diffusion, their ability to use the karyopherin-mediated pathway through the nuclear pore complex (NPC) allows for active import, and subsequent concentration at the INM.

• Rule d — extralumenal domain(s) >~25 kDa. Membrane proteins with extralumenal domains that are >~25 kDa cannot efficiently diffuse across the pore membrane (POM), and localize to the ONM/ER.

• Rule e — extralumenal domain(s) of ~25–75 kDa, with an NLS. The presence of an NLS allows membrane proteins with extralumenal domains between ~25–75 kDa to use the karyopherin-mediated pathway across the POM, but the efficiency of import probably depends on the affinity of the NLS for karyopherin-α42. Therefore, low-affinity NLSs might allow access to the INM, but might not be sufficient to confer exclusive INM localization, resulting in distribution throughout the INM and ONM/ER.

• Rule f — extralumenal domain(s) of ~25–75 kDa with a high-affinity NLS and/or retention. The NLS is sufficient to confer exclusive INM localization owing to a high affinity for karyopherin-α. Alternatively, a membrane protein with a low-affinity NLS could be retained by interacting with elements of the nuclear architecture, such as lamina or chromatin.

Although these rules address the major factors that govern the localization of membrane proteins in the nuclear envelope, additional characteristics of a particular membrane protein will also contribute to its localization. For example, the size of the lumenal domain(s), transmembrane-domain interactions, the capability of membrane proteins to traffic in complexes and the biophysical properties of the hydrophilic and transmembrane domains themselves (such as the electrostatic charge of the extralumenal domain(s)) are probably important.




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AcknowledgementsWe are grateful to R. Peters, S. Wente, R. Wozniak, A. Corbett and L. Veenhoff for insightful discussions regarding INM transport. We thank H. Shi for comments on the initial drafts and M. Rout for valuable criticisms of the manuscript. M.C.K. is supported by a Kirchstein National Research Service Award postdoctoral fellowship and G.B. and C.P.L. are supported by the Howard Hughes Medical Institute.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:UniProtKB: http://ca.expasy.org/sprot

Doa10 | Kapα | Kapβ1 | LAP1 | LBR | MAN1 | Nup2 | NUP50 |

Ydl089w | POM121 | Pom152

FURTHER INFORMATIONPSORTII algorithm: http://www.psort.org

Access to this links box is available online.




highway to the inner nuclear membrane: rules for the road

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