Toward the molecular dissection of protein import into nuclei

397

Toward the molecular dissection of protein import into nuclei Nelly Pant&* and Ueli Aebi fTransport

of proteins,

RNAs

into and out of the nucleus functions

to proceed.

Recent

has led to the identification cytosolic through

factors

exert their function mechanisms

progress

of signals import

However,

at which these this nuclear

import

and of proteins

as the sites on

signals

are still largely unidentified,

underlying

and factors

the molecular

pathway

remain to

be elucidated.

Addresses ‘*ME Miiller Institute for Microscopy, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland *Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 N Wolfe Street, Baltimore, MD 21205, USA *e-mail: [email protected] te-mail: [email protected]

Current 0 Current

Opinion Biology

in Cell Biology Ltd ISSN

1996,

8:397-406

0955-0674

Abbreviations BP CBP GAP GEF hsp70

MDa NE NES NLS NPC NTF2 RCC RNP snRNA snRNP

binding protein cap-binding protein GTPase-activating protein guanine nucleotide exchange

factor

70 kDa heat shock protein megadalton nuclear envelope nuclear export signal nuclear localization sequence nuclear pore complex nuclear transport factor 2 regulator of chromosomal condensation ribonucleoprotein small nuclear RNA small nuclear

the transport of the various ligands different transport pathways.

appears

to occur via

in this area of research

the nuclear

pore complexes.

pore complex

particles

for many cellular

of a number

that mediate

the nuclear

the nuclear

and ribonucleoprotein is essential

RNP

Introduction of proteins, RNAs and riboBidirectional transport nucleoprotein (RNP) particles between the nucleus and the cytoplasm of interphase eukaryotic cells occurs through the nuclear pore complexes (NPCs), -125 megadalton (MDa) supramolecular assemblies embedded in the double-membraned nuclear envelope (NE) (reviewed in [1,2]). Ions, metabolites and small macromolecules can diffuse passively through the NPC via aqueous channels with a physical diameter of -9 nm; larger macromolecules are selectively and actively translocated through a gated channel by a signal-mediated and ATP-dependent mechanism. The ligands which are actively imported through the NPC include nuclear proteins and small nuclear RNP (snRNP) particles, whereas the exported ligands represent small nuclear RNAs (snRNAs), mRNAs, tRNAs and shuttling proteins. Early studies have indicated that

The signals mediating the import of nuclear proteins -called nuclear localization sequences (NLSs) - were identified over two decades ago (reviewed in [Z]). It was subsequently demonstrated that the import of nuclear proteins is a receptor-mediated process. Hence the next step in the study of the nuclear import of NLS-bearing proteins (or NLS proteins) was to identify the receptor recognizing the NLSs. As a consequence, several NLS-binding proteins that interact with NLSs in vitro were identified. However, it was not until the recent development of a digitonin-permeabilized, cytosol-depleted cell system (i.e. one in which the plasma membrane, but not the NE, becomes permeabilized [3]) chat it became possible to demonstrate that of the few NLS-binding proteins identified by in vitro binding assays, only a 54/56 kDa protein purified from bovine erythrocyte cytosol [4] and the 70 kDa heat shock protein (hsp70) [S] are involved in mediated import of NLS proteins. This in vitro transport system has further revealed the requirement of additional cytosolic factors in order to faithfully reconstitute nuclear protein import. Combination of this in vitro protein import assay system with more systematic fractionation of cytosolic extracts has recently led to the identification and characterization of three additional cytosolic factors that mediate the nuclear import of NLS proteins (see below). Moreover, the introduction of an import ligand blot assay [6*] has identified NPC constituents which interact with several of these cytosolic factors in vitro [6’-8’1. Here, we review the progress made over the past 15 months in identifying and characterizing the molecular components that have been implicated in the nuclear import pathway of NLS proteins. In addition, we will briefly present a new perspective on the problem of nuclear export of proteins and RNAs which has emerged recently with the characterization of specific proteins chat rapidly shuttle between the nucleus and the cytoplasm.

NPC structure and chemical composition The NPC is roughly cylindrical in shape with a diameter of -125 nm in the plane of the NE, and it spans the inner and outer membranes of the NE (see Fig. 1). Extensive structural studies (reviewed in [l,Z]) have revealed that the NPC consists of a 52MDa basic framework which is sandwiched between a 32MDa cytoplasmic ring and a 21 MDa nuclear ring. As shown in Figure 1, the cytoplasmic ring is decorated with eight 30-50nm long kinked filaments, whereas the nuclear ring is capped with a basket-like assembly built from eight thin, SO-100nm long filaments joined discally by a 30-50nm diameter

398

Nucleus and gene expression

terminal ring. T h e basic framework of the NPC is made of eight muhidomain 'spokes' embracing a central pore which harbors a - 1 2 M D a 'gated channel' (also called 'plug' or 'transporter') whose definitive structure and exact involvement in mediated transport remain to be more firmly established. Concerning the chemical composition of the NPC, it is built of multiple copies (i.e. 8 or 16 copies, deducible

from the 822 symmetry of the basic framework) of 100 or more different polypeptides called nucleoporins. T h e nucleoporins that have thus far been identified and molecularly characterized represent at most 15% of the entire NPC mass (reviewed in [9,10]). T h e s e nucleoporins exhibit epitopes predominantly found at the cytoplasmic or nuclear periphery of the NPC rather than on its 5 2 M D a basic framework (see Fig. 1; reviewed in [9,10]). Hence, we have to go a long way to more

Figure 1

Schematic diagram summarizing the major structural components of the NPC together with the immunolocalization of five nucleoporin epitopes within the three-dimensional architecture of the NPC. The major structural components of the NPC include the basic framework (i.e. the 'spoke' complex; shown in pink), the central plug or channel .complex (shown in translucent light blue), the cytoplasmic ring and the cytoplasmic filaments (shown in blue), and the nuclear ring and nuclear basket (shown in orange). The central plug or channel complex has been modeled as a translucent ellipsoidal particle to indicate the fact that its definite structure remains elusive. Epitopes of five different nucleoporins are marked in this model: CAN/Nup214/p250, Tpr/p265 and RanBP2/Nup358 exhibit epitopes residing in the cytoplasmic filaments, whereas Nup153 exhibits an epitope near or at the terminal ring of the nuclear basket, and p62 epitopes are located near or at both the cytoplasmic and nuclear faces of the central plug or channel complex. Epitopes for the transmembrane glycoproteins gp210 and POM121 are also displayed in this diagram. On the basis of topological studies, most of the mass of gp210 is predicted to reside in the lumen of the double membrane of the NE (shown in gray). In contrast, most of the mass of POM121 has been localized by immuno-electron microscopy within the NPC proper. Adapted, and reproduced with permission, from [9].

Protein import into nuclei Pant& and Aebi

completely dissect the molecular composition of the NPC. Moreover, as yet relatively little is known about the three-dimensional structure and native conformation of the known nucleoporins, their detailed localization and/or spatial extent within the three-dimensional architecture of the NPC, and their specific interactions with other nucleoporins. This information will, in turn, be key to ultimately deciphering the structural and/or functional involvement of these nucleoporins in nucleocytoplasmic transport. Toward this goal, several nucleoporins have recently been identified which associate with transport factors in vitro (see below).

Nuclear import of proteins through the NPC Nuclear import of proteins is the best characterized nucleocytoplasmic transport activity. Early studies have indicated that this process can be dissected into two distinct steps: first, docking of the nuclear protein destined for import to the NPC periphery; and second, active translocation of the nuclear protein through a gated channel residing in the central pore of the NPC (reviewed in [Z]). The first step does not require nucleotide hydrolysis and is temperature independent, whereas the second step does require nucleotide hydrolysis and is attenuated upon lowering the temperature. Targeting of nuclear proteins to the NPC is specified by short stretches of amino acids called NLSs (reviewed in [ 111). Although the import of the majority of nuclear proteins appears to involve NLSs, as yet unidentified signals mediate the nuclear import of snRNPs (reviewed in [ 121) and of some nuclear glycoproteins [13]. Here we will focus on the nuclear import pathway of NLS proteins. As the ‘NLS receptor’ [4] and several other cytosolic factors that mediate the nuclear import of NLS proteins have been independently identified at the same time by different groups in different species with different techniques, these cytosolic factors have been named differently. To clarify this issue, in Table 1 we have listed the different names for the various homologs of the five cytosolic factors that have been demonstrated to mediate nuclear import of NLS proteins. It is conceivable that additional factors may be involved in the nuclear import of NLS proteins itr viva. Some of these ‘missing’ factors may be associated with the NE or with other cell organelles, and may thereby be retained with the digitonin-permeabilized, cytosol-depleted cells employed to reconstitute nuclear protein import lrl vitro (discussed in [14]). Only four of the five cytosolic factors listed in Table 1 are absolutely required to reconstitute nuclear import of NLS proteins in digitonin-permeabilized cells [6*,15,16**]. These are importin a, importin @. Ran, and nuclear transport factor 2 (NTFZ). Hsp70 probably does not represent an essential transport factor, as it may simply exert its role as a chaperone to facilitate optimal presentation of the NLS so as to mediate the interaction of the NLS with importin a. Moreover, as hsp70 is not required at all for in vitro nuclear import of

399

the glucocorticoid receptor [17], it may simply enhance the targeting efficiency of some nuclear proteins to the cytoplasmic periphery of the NPC. In the next four sections we will discuss the recent studies that have led to the identification of the four cytosolic factors that are necessary and sufficient to reconstitute nuclear import of NLS proteins in vitro.

lmportin a/NLS receptor Importin a-a 60 kDa protein purified from Xenoplls oocytes-was the first cytosolic factor implicated in nuclear import of NLS proteins to be cloned and sequenced [18*]. It binds directly to NLSs and, upon association with importin p (see below), mediates the docking of NLS proteins to the NPC periphery. On the basis of its 60 kDa molecular mass, it was speculated that importin a was the Xenopus homolog of the mammalian NLS receptor (i.e. the 54/56 kDa protein previously purified from bovine erythrocyte cytosol (41). This speculation has recently been confirmed by protein and cDNA sequence analysis of the 54/56 kDa bovine NLS receptor (mentioned in [19*]). Moreover, analysis of the amino acid sequence of importin a has revealed strong homology with a growing family of structurally related proteins that have been cloned and sequenced in unrelated contexts. Members of this family include the yeast SRPl protein which is the product of a gene originally identified as a suppressor of mutants defective in RNA polymerase I [ZO], the human importin a homologs hSRPl/NPI-1, hSRPla and Rchl [16**,21-23,24*], and the most recently identified Drosophih homolog, pendulin/OH031 [25,26]. Consistent with their involvement in nuclear import of NLS proteins, the human homologs of importin a were found by two-hybrid screens through their interactions with NLS proteins. The amino acid sequences of importin a and the other members of this family revealed eight internal repeats of -42 hydrophobic amino acids each; each repeat is called an ‘arm’ motif, a motif originally identified in Armadillo/plakoglobin@catenin proteins [27]. As these proteins link cadherins to the cytoskeleton at intercellular junctions, it is thought that these arm motifs mediate protein-protein interactions [27]. Consistent with this idea, the region of hSRP1 that interacts with the recombination-activating protein RAG-l has been shown to possess four of these arm motifs [Zl]. It has been speculated that the arm motifs of importin a might be involved in binding the NLS and/or in the association of importin a with other cytosolic factors [28*]. Importin a has been shown by immunofluorescence analysis to be present in both the cytoplasm and the nucleus of mammalian cells [28*]. It has, therefore, been suggested that importin a might function as a ‘shuttling carrier’ for NLS proteins that can be recycled to participate in several rounds of transport [4,12]. Recently, it has been more directly demonstrated that importin a does indeed

400

Nucleus and gene expression

Factors required

for the import of nuclear

N8IllO’

other

name(s)

z

protein%

Mammalian

Yeast

homobg(s)t

homolog

hsp701hsc705

FunctIonal

role

Nudeoporin(s)

interacting

with this factor

hsp70

Bnds to NLS proteins.

References

in vitro

t

15.171

Possibly maintains the NLS protein in a transpoe competent conformation. ImportIn cl

lmportin 60

NLS receptor

Kalyopherin a

~541~56

SRPl#/Kap

60

Binds to NLS proteins.

Nupl. Nup2

[416”16’21-2324’26’31 ?>I I,,,

351

PTAc56 hSRPl/NPl-1 hSRP1 a Rchl lmportin 0

lmportin 90

p97lNTF97

Karyopherin 6

pTAC97

Kap95

Mediates docking of the NLS protein-imp&n

Nupll6

16’ II?, 6’ 16” 19’ 30-32

,33’1

a

complex to the NPC. Ran

TC4

RaruTC4

Gspl P, Gsp2p

Acts as a molecular switch

Nup356

136,.3746’

,49” ,651

to commit the transport complex to subsequent translocation steps. B-2

PI0

NTF2

r

May facilitate the transfer

~62

[15.36*1

al the transport complex from the initial docking site to the gated channel. ‘In Xenopus; tbavine. human. mouse and rat homologs; tthese proteins have not yet been identified; gmay not be required for the nuclear unport of all proteins; RDrosophila horn&g pendulin/OH031

is

[25,261.

enter the nucleus concomitantly with the NLS protein, Ran and NTFZ, whereas importin B is retained at the NE ([16**,29**]; see below). It remains to be established whether importin a is involved in the transport steps that follow docking of NLS proteins to the NPC.

lmportin B/p97 A second cytosolic factor-originally identified and purified from bovine erythrocytes and called p97 [30] -is required for the docking of NLS proteins to the NPC. This factor has now been molecularly characterized by several groups and in different species (see Table 1; [6’,8’,19’,31,32,33*]). It is a -97 kDa protein termed importin B [32] that does not reveal any similarity to known proteins. Neither importin a nor importin B . alone is capable of mediating docking of NLS proteins to the NPC, but addition of importin B and importin a together is sufficient to cause accumulation of NLS proteins at the NE of digitonin-permeabilized cells in an ATP-independent and temperature-independent fashion [6*,30-32,33’]. Thus the initial docking of the import ligand complex to the NPC may be mediated by importin B. Consistent with this hypothesis, importin B appears to bind to four mammalian nucleoporins (Nup3.58, Nup214, Nup153 and Nup98) when rat liver NE proteins that have been previously separated by SDS/PAGE are transferred to nitrocellulose filters and probed with W-labeled recombinant importin B [6*,16”]. However, as this ligand blot assay is supplemented with fractionated Xenopus cytosol which contains importin a, it is not yet clear whether the initial docking of the import ligand to the NPC occurs via importin a or importin B.

It has been speculated that as the mammalian nucleoporins that have been found to interact with importin B in the ligand blot assay (described above) contain repetitive XFXFG or GLFG (single-letter code for amino acids) sequence motifs, these act as binding sites for the nuclear import complex [6*,7*,16”]. Unfortunately, as yet the studies that have addressed this speculation are rather contradictory. On the one hand, it was found that the amino-terminal domain of Nup98 (residues 43-518) that contains the repetitive GLFG motif bound NLS proteins in a ligand blot assay [7*]. On the other hand, the domains of the yeast nucleoporins Nup145 and Nup57 containing the repetitive GLFG motif did not bind to the importin a-importin B complex in a solution binding assay [34*]. Moreover, although some nucleoporins may bind to these transport factors in vitro, they might not do so in vtio. For example, it was reported recently that although the three yeast nucleoporins NuplOO, Nupl16 and Nup145 bound Kap95 (the yeast homolog of importin B; see Table 1 and [31]) in a ligand blot assay, only Nupll6 was involved in mediated import of nuclear proteins in viva [8’]. Thus, ligand blot assays may not necessarily be sufficient to document bonnfine functional roles for distinct nucleoporins. In fact, contrary to the claim of Rexach and Blobel [34*], such ligand blot assays may actually be far from an in vitro system that faithfully reproduces nucleocytoplasmic transport. In yeast, genetic and co-immunoprecipitation studies have demonstrated that SRPl (the yeast homolog of importin a) interacts with Nupl and Nup2 [35], and that Kap95 interacts with Nupll6 [8*]. It is thus conceivable that the two transport factors (importin a and importin B) form a

Protein import into nuclei Pant& and Aebi

complex that is recognized by the NPC. In support of this notion, importin B has been shown to bind to the NE of digitonin-permeabilized cells only in the presence of both the NLS protein and importin a [33*]. Unfortunately, we do not yet know the location of the yeast nucleoporins Nupl, Nup2 and Nupl16 within the NPC architecture. It is thus difficult to speculate whether any of these nucleoporins represent, or are part of, the initial docking site for the nuclear import ligand, or whether they only interact with the import ligand complex at a later step of nuclear import. Importin B is shown, by immunofluorescence microscopy, to localize within the cytoplasm, and it also colocalizes with the NE, but it is excluded from the nucleus of intact cells [16**,19*]. In contrast, importin a has been localized in the cytoplasm, to the NE, and in the nucleus [ZB’]. Thus, the complex formed by these two transport factors appears to dissociate at some stage during the interaction with the NPC. Consistent with this hypothesis, it has recently been demonstrated that importin a, but not importin B, enters the nucleus concomitantly with the NLS protein (see Fig. 2; [ 16**,29**]). Interestingly, in immuno-electron microscopy studies, importin B appears to be associated with both the cytoplasmic and nuclear periphery of the NPC [29”], indicating that dissociation of importin B from importin a only occurs after the import ligand complex has traversed the NPC and arrived at its nuclear periphery.

Ran/TC4

and NTFP/plO

Prior to the molecular characterization of importin a and importin 0, two other transport factors that are involved in nucleocytoplasmic transport were identified: first, the small GTPase Ran (or TC4) which has to be in an ‘active’, GTP-bound state to mediate nuclear import of NLS proteins [36,37]; and second, a Ran-interacting protein of 10 kDa termed p10 [15] or NTFZ [38’]. These two transport factors are able to translocate NLS protein complexes that have previously docked to the cytoplasmic NPC periphery into the nucleus of digitonin-permeabilized, cytosol-depleted cells [6’,15], and both of them enter the nucleus concomitantly with the import ligand complex [ 16”]. Ran is an abundant nuclear protein that has been implicated in various nuclear activities (reviewed in [39]). Like other GTPases, Ran acts as a ‘molecular switch’ cycling between an active form, Ran.GTP, and an inactive form, Ran.GDI? Spontaneous conversion between these two states is very slow [40], so it is controlled and accelerated in viva by a variety of regulatory proteins. Three regulators of Ran have been identified so far: first, the nuclear protein RCCl (the regulator of chromosomal condensation) which acts as a guanine nucleotide exchange factor (GEF; reviewed in [39]); second, the cytoplasmic Ran GTPase-activating protein RanGAPl [40-43]; and third, a cytoplasmic Ran-binding protein termed RanBPl which co-activates RanGAPl, and, in the

401

absence of RanGAPl, also inhibits nucleotide exchange on RanGTP [44]. Consistent with the involvement of Ran in nuclear import, mutations in Ran [45], RCCl [46], RanGAPl [43], and RanBPl [47] attenuate or inhibit nuclear import of NLS proteins. Nevertheless, it is as yet unclear how the RanGTPase cycle is coupled with nuclear import of NLS proteins (see Fig. 2). On the one hand, as RanGAPl is a cytoplasmic protein and RCCl, the only known GEF for Ran, is located inside the nucleus, one would expect Ran.GDP to predominate in the cytoplasm. On the other hand, Ran.GTP is required for nuclear import [36,37], so one possibility is that GTP hydrolysis by Ran, and nucleotide exchange at Ran, are confined to the NPC (see below), where some nucleoporin(s) or cytosolic factors might act as regulators of the RanGTPase cycle. The recent discovery of a Ran-binding nucleoporin (RanBPZ/Nup358) has provided additional evidence for a role of Ran in nuclear protein import [48*,49**]. Nup3.58 harbors eight Ran-binding motifs which bind Ran.GTP-but not Ran.GDP--in vitro, and it contains epitopes which localize to the cytoplasmic filaments of the NPC (see Fig. 1; [48*,49”]). Moreover, as an anti-Nup358 antibody inhibited nuclear import of NLS proteins [49”], this Ran-binding nucleoporin appears to play a direct role in NPC-mediated nucleocytoplasmic transport. On the basis of the finding that Ran.GTP-but not Ran.GDP - directly binds to the cytoplasmic filaments of the NPC [48*,50”], it has been proposed that Ran hydrolyzes GTP at the cytoplasmic filaments after both Ran.GTP and the import ligand complex have bound to adjacent sites of a cytoplasmic filament, and that GTP hydrolysis will release the import ligand complex for subsequent transport steps [14,50”]. In support of this proposition, Ran.GTP interacts in vitro with importin B ([34*]; L Gerace, personal communication), and, upon GTP hydrolysis, Ran stimulates dissociation of the NLS protein from the import ligdnd complex [34*]. It is conceivable that additional cycles of GTP hydrolysis by Ran and/or other GTPases -or even ATP hydrolysis by ATPases (see below)-acting at different steps might be required for nuclear import of NLS proteins. Consistent with the hypothesis that other GTPases might be involved in this process, nuclear import of NLS proteins in assays containing a mutant form of Ran (which binds to xanthosine triphosphate rather than GTP) is inhibited by non-hydrolyzable GTP analogs (L Gerace, personal communication). NTFZ, the second cytosolic factor which, together with Ran, is able to translocate the import ligand complex across the central gated channel after it has been docked to the NPC, was identified by its depletion from HeLa cell cytosol with the nucleoporin ~62 [38’]. As ~62 is located at both the cytoplasmic and the nuclear peripheries of the NPC [Sl*‘], near or at the central gated channel, it has been proposed that NTFZ may mediate the delivery of

402

Nucleus and gene expression

Figure 2

Cytoplasm

6 1996 Current Opinion I” Cell Biology representation

with importin a via its NLS. This step takes place in the cytoplasm and does not require physical interaction with any NPC it may, however, be facilitated or accelerated by hsp70. (b) In a second step, this ‘targeting complex’ (consisting of importin

and the NLS protein)

of the import

docks

pathway

to the cytoplasmic

of NLS proteins

periphery

subsequent steps, which result in the transportation of GTP hydrolysis at Ran, and the manner in which

into the nucleus.

(a)

Schematic associates component;

of an NPC by the formation

In the first step, the NLS protein

of a complex

cytoplasm.

It is not yet clear which

importin

a

and importin

a

f3. (c) The

of the NLS protein into the nucleus, require RanGTP and NTFP. However, the exact site the RanGTPase cycle is coupled to the import of NLS proteins, are only poorly understood.

One possibility is that GTP hydrolysis by Ran, and nucleotide exchange at Ran, are confined or cytosolic factors might act as regulators of the RanGTPase cycle. RanGAPl and RanBPl exchange at Ran. After having traversed the central gated channel, the NLS protein, importin nucleus, whereas importin f3 remains associated from importin a, and (e) RanGDP is reactivated

between

to be imported

with the NPC. (d) During or after release by RCCl -catalyzed nucleotide exchange,

form of Ran (i.e. RanGTP

or RanGDP)

the import ligand complex from the initial docking site to the central gated channel [14]. This proposal is supported by the finding that NTFZ, in addition to binding to ~62, also binds Ran-GDP in vifm (L Gerace, persona! communication). Hence NTFZ might act as a regulator of the RanGTPase cycle when Ran is confined to the NPC. For example, after binding to RanGDP, NTFZ might facilitate nucleotide exchange at Ran so that GTP hydrolysis will trigger translocation of the import ligand complex across the central gated channel.

to the NPC (see text) where some nucleoporin(s) may be involved in GTP hydrolysis and nucleotide a, NTF2 and RanGDP are released into the

into the nucleoplasm, the NLS protein dissociates before (f) the transport factors are recycled into the

exists in the cytoplasm

at this point.

Toward the molecular dissection of the N&mediated nuclear import pathway The recent advances made in identifying and characterizing the cytosolic factors that mediate nuclear import of NLS proteins have indicated that, at the molecular level, this process may have to be dissected into more than just docking and translocation steps. As illustrated in Figure 2, in a first step the NLS protein destined for nuclear import binds to importin CLvia its specific NLS. This association takes place in the cytosol, and although it may

Protein import into nuclei Pant6 and Aebi

be facilitated or accelerated by hsp70, it does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks to the cytoplasmic periphery of an NPC by association of importin a with importin p. Whether importin B is already bound to the NPC periphery prior to interacting with the NLS protein/importin a containing targeting complex is, as yet, unclear.

Very little is known about the transport steps that follow importin B mediated docking of the targeting complex to the cytoplasmic periphery of the NPC. As outlined in the previous section, in vitro transport assays indicate that the subsequent steps require Ran.GTP and NTFZ. One scenario suggests that after both Ran.GTP and the import ligand complex have bound to adjacent sites of a cytoplasmic filament, GTP hydrolysis occurs and Ran.GDP now becomes directly associated with the import ligand complex which, upon binding of NTFZ to Ran.GDP, is released from the primary docking site while being delivered to the central gated channel [14].

Another attractive model that has recently been proposed [7’,34*] involves repeated cycles of association and dissociation of the import ligand complex with nucleoporins which may, in turn, drive vectorial translocation of nuclear proteins across the NPC by guided diffusion. This model is based on the in vitro interaction between nucleoporins and NLS proteins in the presence of importin a and importin p that is observed by either a blot overlay assay [7*] or solution binding experiments [34*]. As only nucleoporins that are constituents of peripheral NPC components (i.e. Nup358, Nup214, Nup153, and Nup98; see Fig. 1 and [7’]) were found, by the blot overlay assay, to interact with the import ligand complex [7*], this association-dissociation model may indeed account for the delivery of the import ligand complex from its initial docking site to a second site near or at the cytoplasmic face of the central gated channel. Belanger et a/. [35] also found results that are consistent with the proposed association-dissociation mechanism: Nupl and Nup2, the two yeast nucleoporins used by Rexach and Blobel in their solution binding experiments [34*], bind to SRPl and might, therefore, represent the initial docking site for the import ligand complex [35]. However, as the three-dimensional localization of Nupl and Nup2 within the yeast NPC has yet to be determined, the proposed role of Nupl and Nup2 in nucleocytoplasmic transport remains speculative. Moreover, by a stochastic, guided diffusion type mechanism, translocation of the import ligand complex across the central gated channel (i.e. involving a distance of at least 50nm) would probably be relatively slow, a behavior contrary to what has been observed experimentally (our unpublished data). Last but not least, it is unclear to us how this stochastic association-dissociation mechanism could be vectorial.

403

Yet another elusive aspect of the molecular mechanism underlying NPC-mediated nuclear protein import, which has been traditionally described as an ATP-dependent process, is the role of ATP hydrolysis. The recent demonstration that the small GTPase Ran is directly involved in this energy-requiring transport activity, and that the nuclear import of NLS proteins is inhibited by non-hydrolyzable GTP analogs [36,37], has opened the possibility that GTP-rather than ATP-hydrolysis may be the primary energy source driving NPC-mediated nuclear protein import in vrUo. If so, the requirement for ATP hydrolysis in this process may be more indirect; for example, it may be involved in the recycling of some of the transport factors.

Searching for signals and factors mediating nuclear export The signals mediating import of nuclear proteins (i.e. NLSs) were found in the mid 197Os, but the first export signals have only recently been identified. One reason for this delay has been the indication that the nuclear export of different transport ligands is mediated by class-specific, rather than common, factors. For example, although mRNA competitively inhibits its own export, it does not affect the export of other classes of RNAs [SZ]. As most RNAs are associated with proteins to form RNP particles-which probably represent the bona fine export ligands-a number of RNA-binding proteins that might mediate nuclear export have been identified (reviewed in [53]). Nevertheless, the question has remained as to whether the nuclear signal which commits a particular RNP particle to nuclear export resides on the RNA, on any of the RNA-binding proteins, or on both RNA and its binding proteins. Only recently has it finally been demonstrated that some of these RNA-binding proteins are bonaja’e export factors. These proteins include: firstly, a nuclear cap-binding protein (CBP) complex which is composed of two proteins, CBPZO and CBP80, and which mediates the export of U-rich snRNAs (U snRNAs) which contain a 5’ trimethyl G cap [54**]; and secondly, the HIV-l Rev protein which directs export of unspliced and partially spliced HIV mRNAs [55]. A number of RNA-binding proteins, in addition to proteins that do not directly interact with RNAs, are known to shuttle between the nucleus and the cytoplasm. Molecular characterization of three of these shuttling proteins-namely the heat-stable protein kinase inhibitor (PKI), Rev, and the heterogeneous nuclear RNP (hnRNP) protein Al -has recently led to the identification of short stretches of amino acids, called nuclear export signals (NESS). When fused to heterologous proteins, NESS rapidly translocate these proteins from the nucleus to the cytoplasm ([56**-58”]; reviewed in [59]). The NES of both PKI and Rev consists of a -10 amino acid long leucine-rich core. In contrast. the NES of Al has been

404

Nucleus

and gene

expression

associated with a 3%residue-long region that does not share any sequence similarity with the NESS of PKI and Rev. The absence of a consensus sequence for NESS is consistent with the notion that there exist several distinct nuclear export pathways. Less is known about the cellular factors mediating nuclear export. It is conceivable that some of the same transport factors involved in nuclear import of NLS proteins (see above) might also be involved in at least some of the nuclear export pathways. Thus far, only Ran has been demonstrated to be involved in both nuclear import and export of proteins [60] and in nuclear export of mRNAs [45]. The recent findings on NESS [56”-%**I have now opened the way to search for ‘NES receptors’. In fact, a first candidate for a NES receptor has recently been identified in both human cells [61*,62*] and yeast [63*]. This protein, alternatively termed Rab [61*,62*] and Rip1 [63*], was found by interaction with the Rev activation domain using the yeast two-hybrid system. However, as it yields some homology with nucleoporins, and, at least in yeast, is located at the NE (in human cells it is a nuclear protein), it remains to be determined whether Rab/Ripl is a botlnfine nucleoporin or a soluble transport factor. Moreover, it remains to be elucidated whether the interaction between Rab/Ripl and the Rev activation domain does actually involve the NES of Rev.

import, individual nucleoporins are marked by antibodies caged with another size of colloidal gold particle. With the same goal in mind, similar experiments are under way to follow the nuclear export of different RNAs.

Acknowledgements WCwouldlike to thank Christian

Hcnn and IIanicl Stofflcr for dcsianing and preparing I:igurc 1, and Ronald A MilliRan (Scripps Kcscarch Institute, La Jolla) for providing the three-dimensional data set used by C Hcnn and 11 Stofflcr to produce this figure. ‘I’his work was supported by the Kanton Ilascl-Stadc, the ME hliillcr Foundation of Switzerland, and by rcscarch grants from the Swiss National Science I~oundation (SNI:) and rhc Human Frontier Scicncc Program Organization (HI:SI’O).

References

. l

To eventually get a complete molecular picture of the nuclear import pathway of NLS proteins, we obviously have to go beyond ligand blot assays with nitrocellulose-immobilized putative transport factors or nucleoporins-after all, ‘seeing is believing’. For this we and others have started to follow the purpose, spatial and temporal fate of microinjected colloidal gold labeled NLS proteins during their translocation from the cytoplasm into the nucleus by electron microscopy [64]. To colocalize distinct transport factors, these factors are tagged with gold particles of different sizes and co-injected with the labeled NLS protein. Moreover, to molecularly identify the site(s) of interaction of the NLS protein and a particular transport factor with the NPC during nuclear

reading

of special interest

*

of

outstanding interest

1.

PantC! N, Aebi U: Towards understanding the 3-D structure of the nuclear pore complex at the molecular level. Curr Opin Srrucf f3iol 1994, 4:167-l 96.

2.

Pant6 N, Aebi U: Toward a molecular understanding of the structure and function of the nuclear pore complex. Int Rev Cytol 1995, 1628:225-255.

3.

Adam SA, Steme-Marr R, Gerace L: Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol 1990, 111:807-616.

4.

Adam SA, Gerace L: Cytosolic proteins that specifically bind nuclear localization signals are receptors for nuclear import Cell 1991, 66:837-647.

5.

Shi Y. Thomas JO: The transoort of oroteins into the nuc&s requires the 70-kilddalton heat shock protein or its cytoplasmic cognate. MO/ Cell Biol 1992, 12:2186-2192.

Conclusions Dissecting the molecular details underlying nuclear import of NLS-bearing proteins through the NPC has only just begun. A major reason for this relatively slow progress has been the difficulty in identifying and molecularly characterizing the transport factors mediating this process. Furthermore, the sites on the NPC at which these factors exert their action have to be identified molecularly. With the molecular architecture of the NPC slowly but definitely shaping up (see above and Fig. l), the time has now come to more systematically identify and characterize in space and time the transport factors and nucleoporins which sequentially-or repeatedly-interact with NLS proteins as these move along their nuclear import pathway.

and recommended

Papers of particular interest, published within the annual period of review, have been highlighted as:

6. .

Radu A, Blobel G, Moore MS: Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc Nat/ Acad Sci USA 1995, 92:1769-l 773. By employing a ligand blot assay with nitrocellulose-immobilized rat liver nuclear envelope proteins, a 9s Xenopus ovary cylosolic protein complex, termed karyopherin, has been identified and characterized. Karyopherin consists of the three karyopherin subunits al (54 kDa), a2 (56 kDa), and B (97 kDa) (see Table l), and it mediates the docking of nuclear import ligands to the NPC periphery via nucleoporins Nup358 (see [48*,49**]), Nup214, Nupl53, and Nup98 (see [7*1). The rat homolog of karyopherin b has also been molecularly cloned and sequenced (reported in [6*]). 7. .

Radu A, Moore MS, Blobel G: The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell 1995, El:21 5-222. Nup98, which is shown by immuno-electron microscopy to be asymmetrically located at the nuclear NPC periphery, functions as one of several docking site nucleoporins in karyopherin-mediated binding of nuclear import ligands (see [So]). The cDNA-deduced primary structure of this O-linked, wheat germ agglutinin reactive glycoprotein harbors numerous repetitive GLFG (singleletter code for amino acids) and FG sequence motifs and some XFXFG sequence repeats in its amino-terminal half, so it represents a vertebrate member of a family of yeast GLFG nucleoporins (see @*I). On the basis of ligand blot essays, it is suggested that the amino-terminal peptide repeat domain of Nup98 represents a karyopherin-mediated docking site for import ligands. 8. .

lovine MK, Watkins JL, Wente SR: The GLFG repetitive region of the nucleoporin Nupll6p interacts with Kap95p, an essential yeast nuclear import factor. J Cell f3iol 1995, 131 :1699-l 713. Three approaches have been taken to dissect the structural basis for Nupl 16’s role in yeast nuclear import: first, deletion mutagenesis analysis reveals that Nupl 16’s GLFG (single-letter code for amino acids) repeat domain is required for NPC function; second, overexpression of Nupl 16’s GLFG repeat domain severely inhibits cell growth and rapidly blocks polyadenylated-RNA export; and third, biochemical and two-hybrid analyses characterized an interaction between Nupl16’s GLFG repeat domain and Kap95p, the yeast homolog of vertebrate karyopherin fi (see IS*]). 9.

Pant6 N, Aebi U: Toward the molecular details of the nuclear pore complex. J Sfruct Biol 1994, 113:179-l 89.

Protein import into nuclei Pante and Aebi

10.

Bastes R, Pante N, Burke B: Nuclear pore complex proteins Rev Cyt 1995, 16281257-302.

11.

Boulikas T: Nuclear localization Gene Expr 1993,3:193-227.

12.

Gerace L: Molecular trafficking across the nuclear pore complex. Curr Opin Cell Biol 1992, 41637-645.

13.

Duverger E, Pellerin-Mendes C, Mayer R, Roche A-C, Monsigny M: Nuclear import of Qlycoconjugates Is distinct from the classical NLS pathway. J Cell Sci 1995,108:1325-1332.

14.

Melchior F, Gerace G: Mechanism of nuclear protein Import. Curr Opin Cell Bioll995, 7~31 O-31 8.

15.

Moore MS, Blobel G: Purification of a Ran-interacting protein that Is required for protein Import into the nucleus. Proc Nat/ Acad Sci USA 1994, 91:10212-10216.

lnt

signals (NLS). Crit Rev Eukar

Moroianu J, Hijikata M, Blobel G, Radu A: Mammalian keryopherin aI8 and a*5 heterodimers: at or a2 subunit binds nuclear localization slgnal and 5 subunit interacts with peptide repeat-containing nucleoporins. Proc Nat/ Acad Sci USA 1995, 92:6532-6536. Describes the functional characterization of Rchl, one of the human homologs of importin a. The localization of importin a, importin 5, Ran and NTF2 at 30 minutes after their addition to digitonin-permeabilized, cytosol-depleted cells demonstrated that importin a, Ran and NTFP enter the nucleus concomitantly with the NLS protein, whereas importin 5 is retained at the NE. 16. ..

1 7.

Yang J, DeFranco DB: Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen. MO/ Cell Biol 1994, 14:5088-5098.

26. .

lmamoto N, Shimamoto T, Takao T, Tachibana T, Kose S, Matsubae M, Sekimoto T, Shimonishi Y, Yoneda Y: In viva evidence for involvement of a 58 kDa component of nuclear pore-targetlng complex in nuclear protein import EMBO J 1995,14: 3617-3626. By molecularly cloning and sequencing a 56 kDa protein of the nuclear pore targeting complex, it has been shown that this protein, termed PTAC58 or m-importin, represents the mouse homolog of Xenopus karyopherin a (see Table 1 and [6*1). On the basis of results obtained from the cytoplasmic injection of antibodies raised against recombinant m-importin, evidence has bean provided that m-importin is involved in nuclear protein import through association of m-importin with the NLS of the import ligand prior to NPC binding. 29. ..

Giirlich D, Vogel F, Mills AD, Hartmann E, Laskey R: Distinct functions for the two importin subunits in nuclear protein import Nature 1995, 377:246-248. Detection of the NLS protein, importin a or importin f3 at 30 minutes after their addition to digitonin-pemreabilized HeLa cells demonstrated that importin a enters the nucleus concomitantly with the NLS protein, whereas importin 5 is confined to the NE. By using immunogold electron microscopy, importin 5 was found to be associated with both the cytoplasmic and the nuclear peripheries of the NFC in rat liver cryosections. 30.

Adam EJH, Adam SA: Identification of cytosolic factors required for nuclear localization sequence-mediated binding to the nuclear envelope. J Cell B/o/ 1994, 125:547-555.

31.

Enenkel C, Blobel G, Rexach M: Identtficatton of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes. J B/o/ Chem 1995, 270:16499-l 6502.

32.

Gorlich D, Kostka S, Kraft R, Dingwall C, Laskey R, Hartmann E: Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Gun B/o/ 1995, 5:383-392.

16. .

Giirlich D, Prehn S, Laskey R, Hartmann E: Isolation of a protein that is essential for the first step of nuclear protein import Cell 1 QQqv 79:767-778. Describes the first cytosolic transport factor that was molecularly characterized. The gene encoding importin a, the Xenopus homolog of the mammalian NLS receptor [4], was isolated, cloned and sequenced. Both the isolated and the recombinant protein were able to dock NLS proteins to the NE of digitonin-permeabilized HeLa cells. 19. .

Chi NC, Adam EIH, Adam SA: Sequence and characterization of cytoplasmic nuclear protein import factor p97. J Cell Biol 1995, 130:265-274. The gene encoding the cytosolic transport factor ~97, a protein previously identified and purified from bovine erythrocytes I301, was cloned, sequenced and identified as the mammalian homolog of Xenopus importin 5.. By immunofluorescence microscopy, p97 was localized to the cytoplasm and the NE of mammalian cells. An anti-p97 antibody inhibited the import of NLS proteins into nuclei of digitonin-permeabilized, cytosol-depleted cells, but did not inhibit the binding of the import kgand complex to the NPC. 20.

Yano R, Oakes M, Yamaghishi M, Dodd JA, Nomura M: Cloning and characterization of SRPI, a suppressor of temperaturesensitive RNA polymerase I mutations, in Seccharomyces cerevisiee. MO/ Cell B/o/ 1992, 12:5640-5641.

21.

C&es P, Ye Z, Baltimore D: RAG-1 interacts with the repeated amino acid motif of the human homologue of the yeast protein SRPl. Proc Nat/ Acad Sci USA 1994, 91~7633-7637.

22.

Cuomo CA, Kirch SA, Gyuris J, Brent R, Oettinger MA: Rchl, a protein that specifically interacts with the RAG-l recombination-activating protein. Proc Nat/ Acad Sci USA 1994, 91:6156-6160.

23.

O’Neill RE, Palese P: NPI-1, the human homolog of SRP-I, interacts with influenza virus nucleoprotein. l’/ro/ogy 1995, 206:116-l 25.

Weis K, Mattaj IW, Lamond Al: Identification of hSRPla as a functional receptor for nuclear localization sequences. Science 1995,26&l 049-l 053. hSRPla, a human homolog of importin a, was found in a yeast twohybrid screen. A 90 kDa protein (importin 5) was co-immunoprecipitated with hSRP1 a. It was demonstrated by immunoprecipitation that hSRP1 a can directly associate with NLSs.

33. .

lmamoto N, Shimsmoto, T, Kose S, Takao T, Tachibana T, Matsubae M, Sekimoto T, Shimonishi Y, Yoneda Y: The nuclear pore-targeting complex binds to nuclear pores after association with a karyophile. FEBS Left 1995, 368:415-419. In this study, a 97 kDa protein of the nuclear pore targeting complex, termed PTACQ7, has been molecularly cloned and sequenced. PTA97 represents the mouse homolog of Xenopus karyopherin f3 (see Table 1 and [So]). Biochemical analysis has revealed that PTAC97, in a 1 : 1 molar complex with PTAC58 (which binds to karyophilic proteins; see [28*]), completely reconstitutes docking of nuclear import ligands to the NPC (see also [6-l). 34. .

Rexach M, Blobel G: Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporlns. Cell 1995, 83:683-692. In this in vitro study, the molecular dynamics of nuclear protein import have been examined by a solution binding assay involving an NLS-bearing ligand, the transport factors karyopherin a and karyopherin 8, the small GTPase Pan, and FXFG or GLFG (single-letter code for amino acids) repeat domains of yeast nucleoporins. On the basis of the resulting in vitro binding reactions, it is proposed that translocation of NLS proteins across the NPC is a stochastic process that operates via repeated association-dissociation reactions of the import ligand complex with the FXFG repeat domains of distinct nucleoporins lining the translocation pathway (see also [6’,7*]). 35.

Belanger KD, Kenna MA, Wei S, Davis L: Genetic and physical interactions between Srpl p and nuclear pore complex proteins NUPl p and NUPZp. J Cell Biol1994, 126:61 g-630.

36.

Moore MS, Blobel G: The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 1993, 365:661-663.

37.

Melchior F, Paschal B, Evans J, Gerace L: Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol 1993, 123:1649-l 659.

24. .

25.

26.

27

KiJssel P, Frasch M: Pendulin, a Drosophile protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation. J Cell Bioll995, 129:1491-l 507. Torijk I, Strand D, Schmitt R, Tick G, Tarok T, Kiss I, Mechler M: The overgrown hemetopoietic orgens-31 tumor suppressor gene of Drosophi/8 encodes an importin-like protein accumulating in the nucleus at the onset of mitosis. J Cell B/o/ 1995, 129:1473-l 489. Peifer M, Berg S, Reynolds AB: A repeating amino acid motif shared by proteins with diverse cellular roles. Cell 1994, 76:769-791.

405

38. .

Paschal BM, Gerace L: Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein ~62. J Cell B/o/ 1995, 129:925-937. Describes the purification and cloning of the transport factor NTF2, from HeLa cell cytosol, via interaction with the nucleoporin ~62.

39.

Dasso M: RCCl in the cell cycle: the regulator of chromosome condensation takes new roles. Trends Biochem Sci 1993, 18:96-l 01.

40.

Bischoff FR, Klebe C, Kretschmer J, Wittinghofer A, Ponstingl H: RanGAPl induces GTPase activity of nuclear Ras-related Ran. Proc Nat/ Acad SC/ USA 1994, 91:2587-2591.

41.

Becker J, Melchior F, Gerke V, Bischoff FR, Ponstingl H, Wittinghofer A: RNA1 encodes a GTPase-activating protein specific for Gspl p, the RanfTC4 homologue of S8cch8romyces cefevisiae. J t3iol Chem 1995. 270:11860-l 1865.

406

Nucleus

and gene expression

42.

Bischofl FR, Krebber H, Kempf T, Hermes I, Ponstingl H: Human RanGTPase-activating protein RanGAPl is a homologue of yeast Rnal p involved In mRNA processing and transport. Proc Nat/ Acad Sci USA 1995, 92:1749-l 753.

52.

Jannolowski A, Boelens WC, lzaurralde E, Mattaj IW: Nuclear export of different classes of RNA Is mediated by specific factors. J Cell Biol. 1994, 124:827-835.

53.

lzaurralde E, Mattaj IW: RNA export

43.

Corbett A, Koepp DM, Schlenstedt G, Lee MS, Hopper AK, Silver P: Rnal p, a RanITC4 GTPase activating protein, is required for nuclear import. J Cell Biol 1995, 130:1017-l 026.

54. ..

44.

Bischoff FR, Krebber H, Smirnova E, Dong W, Ponstingl H: Coactivation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBPl. EM60 J 1995, 14:705-715.

45.

Schlenstedt G, Saavedra C, Loeb JDJ, Cole CN, Silver P: The GTP-bound form of the yeast RanITC4 homologue blocks nuclear protein import and appearance of poly(A)+RNA in the cytoplasm. Proc Nat/ Acad Sci USA 1995, 92:225-229.

46.

Tachibana T, lmamoto N, Seino H, Nishimoto T, Yoneda Y: Loss of RCCl leads to suppression of nuclear protein import in living cells. Biol Chem 1994, 269:24542-24545.

4-Z

Schlenstedt G, Wong DH, Koepp DM, Silver P: Mutants in a yeast Ran binding protein are defective in nuclear transport EMBO J 1995, 14:5367-5378.

46. .

Wu J, Matunis MJ, Kraemer D, Blobel G, Coutavas E: Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region. J Biol Chem 1995, 270:14209-l 4213. - --By screening a HeLa cell expresslon library wth recombinant Kan.ti I P, a protein with a calculated molecular mass of 358 kDa has been molecularly cloned and sequenced. It has been localized at or near the tip of the cytoplasmic filaments of NPCs by immuno-electron microscopy (using colloidal gold-labeled antibodies raised against a 288 amino acid segment expressed in Escherichia co/r), and as a result of this localization it has been identified as a nucleoporin and named Nup358. With its four RamGTP-binding sites and its FXFG (single-letter code for amino acids) repeats, Nup358 is designated a nucleoporin that contains binding sites for two transport factors, namely karyopherin B and Ran.GTP. Hence it may be involved in the initial docking of nuclear import ligand complexes to the cytoplasmic periphery of the NPC (see also [49**,50**1). 49. ..

Yokoyama N, Hayashi N, Seki T, Panti? N, Ohba T, Nishii K, Kuma K, Hayashida T, Miyata T, Aebi U et al: RanBP2, a giant nucleopore protein which binds Ran/TC4. Nature 1995, 376:184-l 88. Using a two-hybrid screen with human Ran cDNA as a ‘bait: human RanBPP has been molecularly cloned and characterized. On the basis of its deduced 2 224 residue long amino acid sequence, RanBP2 appears to be identical to human Nup358 (see [48*]). By using immunoelectron microscopy it is shown that RanBPP exhibits epitopes at the cytoplasmic filaments of the NPC. The finding that the nuclear import of NLS proteins is inhibited by an antibody directed against RanBP2 suggests that this giant nucleoporin plays a role in NPC-mediated nucleocytoplasmic transport. Melchior F, Guan T, Yokoyama N, Nishimoto T, Gerace L: GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import I Cell Viol 1995, 131:571-581. By light and electron microscope immunolocalization, the small GTPase Ran is shown to accumulate at the cytoplasmic periphery of the NPC in the presence of non-hydrolyzable GTP analogs which inhibit nuclear import of NLS-bearing ligands. A single prominent polypeptide corresponding to the 358 kDa nucleoporin Nup358/RanBP2 has been detected by blot overlay of isolated rat liver nuclear envelopes with Ran.GTP (see [48’,49”1). On the basis of these findings, it is proposed that RanBPP is likely to constitute the Ran.GTP-binding site identified at the cytoplasmic periphery of the NPC, thus supporting a model in which initial import ligand binding to the NPC occurs at or near RanBP2, and hydrolysis of GTP by Ran at this site serves to commit the import ligand complex to the nuclear import pathway.

50. ..

Guan T, Mijller S, Kleir G, Pant6 N, Blevitt JM, Hlner M, Paschal B, Aebi U, Gerace L: Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex. MO/ Biol Cell 1995, 6:1591-l 603. The p62 complex, which interacts with cytosolic transport factors and which represents part of the nucleocytoplasmic transport machinery of the NPC, has been purified from rat liver nuclei. It consists of four distinct O-linked glycoproteins (~62, ~58, p54 and ~45) in an equimolar complex which has a mass of -234 kDa and a pronounced tendency to oligomerize into larger particles. By electron microscopy it appears as a donut-shaped particle with a diameter of -15 nm.

51. ..

Cell 1995, 81 :153-l

59.

lzaurralde E, Lewis J, Gamberi C, Jarmolowski A, McGuigan C, Mattaj IW: A cap-binding protein complex mediating U snRNA export Nature 1995, 376:709-712. Evidence is provided for a role of the nuclear cap binding protein complex (which is composed of the two CBPs CBPEO and CBP20) in U-rich snRNA nuclear export. Microinjection of an antiCBP20 antibody into Xenopus oocytes results in an inhibition of nuclear export of U snRNAs. 55.

Fisher U, Meyer S, Teufel M, Heckel C, Lijhnnann R, Rautmann G: Evidence that HIV-1 Rev directly promotes the nuclear export of unspliced RNA. EMBO J 1994, 13:4105-4112.

56. ..

Wen W, Meinkoth JL, Tsien RY, Taylor SS: Identification of a signal for rapid export of proteins from the nucleus. Cell 1995, 82~463-473. The NES of the heat-stable protein kinase inhibitor that promotes export of the catalytic subunit of CAMP-dependent protein kinase has been identified. It consists of a 10 amino acid long leucine-rich core similar to that of the NES of Rev 157*1. By site-directed mutagenesis it was shown that three of the four leucine residues and an isoleucine are important for nuclear export. 57. ..

Fischer U, Huber J, Boelens WC, Mattaj IW, Liihrmann R: The HIV-1 Rev activation domain is a nuclear exoort sianal that accesses an export pathway used by speck cell~ar RNAs. Cell 1995. 821475-483. The nuclear expori activity of Rev was identified within a 9 amino acid long leucine-rich peptide of the activation domain of Rev. Bovine serum albumin (BSA) conjugated with the NES of Rev (termed BSA-R) inhibited Revmediated nuclear export of RNAs containing the Rev response element. By co-injection of BSA-R with different RNAs into Xenopus oocfles, it was shown that BSA-R competes for the export of 5s rRNA and U snRNAs (but not for the export of mRNAs, tRNAs and ribosomal subunits). Hence it was concluded that the export pathway of Rev is also used by other RNAs. 58. ..

Michael WM, Choi M, Dreyfuss G: A nuclear export signal in hnRNP Al: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell 1995, 83:415-422. The signal in the heterogeneous nuclear RNP (hnRNP) protein Al that promotes nuclear export of this protein was identified within a 38 amino acid long domain (termed M9); this domain also contains the unique NLS of Al. By site-directed mutagenesis it was shown that specific amino acids within the M9 domain of the Al protein are critical for the nuclear export of Al. 59.

Gerace L: Nuclear export signals and the fast track to the cytoplasm. Cell 1995, 82:341-344.

60.

Moroianu J, Blobel G: Protein export from the nucleus requires the GTPase Ran and GTP hydrolysis. froc Nat/ Acad Sci USA 1995,92:4318-4322.

61. .

Bogerd HP, Fridell RA, Madore S, Cullen BR: Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell 1995, 82:485-494. A human Rev activation domain binding protein (termed Rab; see also [62*]) that is a candidate for an NES receptor was identified in a yeast two-hybrid screen. This protein functionally interacts with the Rev activation domain when Rev is bound to its RNA target. 62. .

Fritz CC, Zapp ML, Green MR: A human nucleoporin-like protein that specifically interacts with HIV Rev. Nature 1995, 376:530-533. The same human Rev activation domain binding protein as described in [61’], tened Rab, was identified by the authors of this paper. This protein was present in nuclear extracts of HeLa cells, and was localized at the NE and within the nucleoplasm of HeLa cells by immunofluoresence microscopy. 63. .

Stutz F, Neville M, Rosbash M: Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast Cell 1995, 82:495-506. The yeast homolog of Rab [61*,62*], termed Ripl, was identified in a yeast two-hybrid screen, and it was localized to the NE of yeast cells by immunofluoresence microscopy. Both overexpression of the gene encoding this protein and gene disruption reduced the activity of Rev in transactivation assays. 64.

Pante?N, Aebi U: Exploring nuclear pore complex structure and function in molecular detail. J Cell Sci 1995, 1 OE(suppl 19):1-l 1.

65.

Belhumeur P, Lee A, Tam R, DiPaolo T, For-tin N, Clark MW: GSPl and GSP2, genetic supressors of the prp20-I mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization. MO/ Cell Biol 1993, 13:2152-2161.