COMMENTARY
3233
Targeting of Ran: variation on a common theme?
Markus Künzler* and Ed Hurt
Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, 4. OG, Heidelberg 69120, Germany
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science 114, 3233-3241 (2001) © The Company of Biologists Ltd
Summary
The Ran GTPase plays a key role in nucleocytoplasmic
transport. In its GTP-bound form, it directly interacts with
members of the importin β family of nuclear transport
receptors and modulates their association with cargo.
Work in cell-free higher-eukaryote systems has
demonstrated additional roles for Ran in spindle and
nuclear envelope formation during mitosis. However, until
recently, no Ran-target proteins in these cellular processes
were known. Several groups have now identified importin
β as one important target of Ran during mitotic spindle
formation. This finding suggests that Ran uses the same
effectors to regulate different cellular processes.
Introduction
Small GTPases of the Ras superfamily regulate many cellular
processes, including growth, morphogenesis, cell motility,
axonal guidance, cytokinesis, and trafficking through the
Golgi, endosomes and nucleus (Takai et al., 2001). Because of
their low intrinsic GTP hydrolysis and guanine-nucleotideexchange activity, these proteins exist in two states, GTP bound
and GDP bound, whose interconversion is regulated by
GTPase-activating proteins (GAPs), guanine-nucleotide
exchange factors (GEFs) and GDP-dissociation inhibitors
(GDIs). The GTPases act as molecular switches, in which the
‘on’ or ‘active’ state is the GTP-bound form. The GTP-bound
state interacts with targets or effectors that link the switch to
specific cellular processes.
The small Ras-like GTPase Ran, one of the most highly
conserved proteins in eukaryotes, is essential for cell viability
in all organisms tested (Macara et al., 2000). Alterations in
the Ran GTPase cycle produce a very pleiotropic phenotype.
Whether this reflects functions of Ran in multiple cellular
processes or merely its pivotal role in nucleocytoplasmic
transport has been debated for some time. The recent
demonstration that Ran functions in mitotic spindle and
nuclear envelope formation independently of its role in
nucleocytoplasmic transport (Hetzer et al., 2000; Sazer and
Dasso, 2000; Zhang and Clarke, 2000; Zhang and Clarke,
2001) argues for the former explanation. However, in contrast
to the role of Ran in nucleocytoplasmic transport, the
molecular mechanisms of these additional functions of Ran
are poorly understood. Here, we give an overview of the
proteins in higher and lower eukaryotes that are known to
bind to the two nucleotide-bound states of Ran. We
hypothesize that the importin-β-like family of nuclear
transport receptors represents, in a strict sense, the only Ran
target known to date. Interestingly, recent evidence
demonstrates that this family of proteins at least partially
underpins the function of Ran in mitotic spindle formation as
well as its crucial role in nucleocytoplasmic transport. This
suggests that the same class of proteins can link a given
GTPase to different cellular processes (Dasso, 2001;
Melchior, 2001).
The Ran GTPase cycle
In contrast to other members of the Ras GTPase superfamily,
Ran is a very abundant, soluble and predominantly nuclear
protein (Macara et al., 2000). A crucial feature of the Ran
GTPase cycle is the spatial separation of GTP hydrolysis and
guanine-nucleotide exchange by the nuclear envelope (NE).
This separation is a consequence of the different subcellular
localizations of the known Ran-specific GAP and GEF.
The only known RanGAP, RanGAP1 in higher eukaryotes
or Rna1p in yeast, is a crescent-shape protein, formed by
eleven leucine-rich repeats, that bears no resemblance to
RasGAP or RhoGAP (Hillig et al., 1999) and localizes
exclusively to the cytosol. However, a pool of RanGAP1 is
associated with Nup358/RanBP2, a component of the
cytoplasmic filaments of the mammalian nuclear pore complex
(NPC; Mahajan et al., 1998; Matunis et al., 1998; Saitoh et al.,
1998). This association depends on the post-translational
modification of RanGAP1 by a ubiquitin-related protein,
SUMO-1. Rna1p lacks the domain of RanGAP1 that is
modified and there is no evidence for a modification of this
kind. However, there is evidence that Rna1p is associated with
NPCs (Allen et al., 2001).
Although the association of RanGAP1 with RanBP2 appears
to be important for nucleocytoplasmic transport in some
permeabilized cell systems, the facts that yeast and flies do not
contain a RanBP2 homologue and that RanBP2 in mammals is
not ubiquitously expressed argue against a general role in
nucleocytoplasmic transport. Interestingly, both RanGAP1 and
Rna1p have been proposed to shuttle between the nucleus and
cytoplasm (Feng et al., 1999; Matunis et al., 1998), which
might be an alternative explanation for their association with
NPCs. RanGAP1 has also been found at the mitotic spindle
(Matunis et al., 1996), which might be relevant in the context
of Ran’s role during NE assembly after mitosis (see below).
The only known RanGEF, RCC1 (for regulator of
chromosome condensation 1) in higher eukaryotes and Prp20p
in yeast, is a strictly nuclear protein that appears to be
associated with chromatin (Seki et al., 1996). Recent data
suggest that RCC1 is anchored on DNA by histones H2A and
H2B (Nemergut et al., 2001). However, Fontoura et al. have
Key words: Ran-binding, GSP1, SPI1, Karyopherins, Microtubules
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JOURNAL OF CELL SCIENCE 114 (18)
recently reported a pool of RCC1 at the NPC, suggesting that
RCC1 has alternative localizations (Fontoura et al., 2000).
RCC1 has a highly repetitive primary structure, exhibiting a
seven-bladed β-propeller fold similar to that of the β-subunits
of heterotrimeric G proteins (Renault et al., 1998), and the
recent crystal structure of the RCC1-Ran complex confirms
previous suggestions that Ran binds to one face of the propeller
(Renault et al., 2001). RanGEF interacts mainly with the
switch II region of Ran, one of two highly flexible regions of
the conserved nucleotide-binding fold of Ras-like
GTPases that undergo major conformational
changes upon transition between the two nucleotidebound states (Bourne et al., 1991).
The compartmentalization of RanGAP and
RanGEF has two important consequences. First, Ran
has to shuttle between the cytoplasm and
nucleoplasm in order to complete one round of GTP
hydrolysis and guanine-nucleotide exchange. This is
evident from the altered steady-state localization of
Saccharomyces cerevisiae Ran, Gsp1p, in cells
carrying loss-of-function mutations in the genes
encoding the yeast homologues of RanGAP1 (rna11) and RCC1 (prp20-1). The predominantly nuclear
signal of Gsp1p in wild-type cells is enhanced in the
rna1-1 mutant but not in the prp20-1 mutant (Fig. 1;
Stochaj et al., 2000). The growth defect of a prp201 mutant at restrictive temperature can be partially
suppressed by overexpression of Gsp1p, which
suggests that the nuclear concentration of Ran is
critical for yeast cell viability (Belhumeur et al.,
1993; Kadowaki et al., 1993). Second, the two
nucleotide-bound states of Ran are asymmetrically
distributed with respect to the NE: the predominant
form in the cytoplasm is RanGDP, whereas the
predominant form in the nucleoplasm is RanGTP.
This steep RanGTP gradient across the nuclear
envelope is thought to impart directionality on
nucleocytoplasmic transport (see below).
Ran targets
NTF2 and related proteins
NTF2 (nuclear transport factor 2; p10) was identified
with Ran in a biochemical fraction required for
active nuclear import of a model substrate in
digitonin-permeabilized cells (Moore and Blobel,
1994). The protein was shown to play an essential
role in replenishing the nucleus with Ran by acting
as a nuclear import receptor for Ran both in
permeabilized cell systems (Ribbeck et al., 1998;
Smith et al., 1998; Steggerda et al., 2000) and in vivo
(Quimby et al., 2000a; Quimby et al., 2000b). NTF2,
which has a molecular weight of only 10 kDa, forms
a homodimer and binds directly and specifically to
two molecules of Ran in its GDP-bound form. The
crystal structure of the protein complex reveals
direct contacts between the hydrophobic cavity of
NTF2 and the switch II region of RanGDP (Stewart
et al., 1998). Independently of its binding to
RanGDP, NTF2 interacts with FG-repeat-containing
nucleoporins (Clarkson et al., 1996; Paschal and
Gerace, 1995) and localizes to NPCs at steady state (M.K. and
E.H., unpublished). The interactions between NTF2 and both
RanGDP and FG repeats are essential for nuclear import
(Bayliss et al., 1999; Clarkson et al., 1997; Paschal et al., 1997;
Wong et al., 1997). The overlap of NTF2-binding sites on the
NPC with those for the prototypical nuclear transport receptor,
importin β, might explain the observed interference between
NTF2 and the classical nuclear import route, which is mediated
Fig. 1. (A) The Ran GTPase cycle. GTP hydrolyis and guanine-nucleotide
exchange on Ran occur in the cytoplasm and the nucleus, respectively, owing to
the localization of the only known Ran-specific GTPase-activating protein (GAP)
and guanine-nucleotide-exchange factor (GEF). Additional Ran-binding proteins,
RanBP1 and Mog1, coactivate the reactions as indicated. Nuclear import and
export of Ran through the nuclear pore complexes (NPCs, shown in grey) are
accomplished by the RanGDP-binding NTF2 protein and the family of RanGTPbinding karyopherins (Imp/Exp), respectively. (B) Dynamic steady-state
localization of Ran (Gsp1p) visualized in S. cerevisiae. A low-copy number
plasmid encoding GFP-Gsp1p under control of the NOP1 promoter (Lau et al.,
2000) was introduced into temperature-sensitive S. cerevisiae strains defective for
RanGAP (rna1-1) and RanGEF (prp20-1) and an isogenic wild-type strain.
Fluorescence microscopy was performed as described (Lau et al., 2000).
Targeting of Ran: variation on a common theme?
3235
Table 1. Known Ran-binding proteins
Protein class (S. cerevisiae homolog)
Binding specificity (experimental evidence)
Steady-state localization
RanGAP1 (Rna1p)
RCC1 (Prp20p)
NTF2/p10/pp15 (NTF2)
NXT1/p15 (na)
Nup358/RanBP2 (na); Nup153 (na)
RanGTP (iv)
RanGDP, RanGTP, empty Ran (iv)
RanGDP (iv)
RanGTP (iv)
RanGDP (iv)
Mog1 (Mog1p)
RanGDP, RanGTP, empty Ran (iv)
Cytoplasm and NPC
Nucleus and NPC
NPC
Nucleus and NPC
Cytoplasmic and nuclear face
of NPC, respectively
Nucleus
Dis3 (Dis3p)
RanGDP, RanGTP,empty Ran (iv)
Nucleolus
Karyopherins
RanGTP (iv)
Nucleus, cytoplasm, NPC
RanBP1 (Yrb1p), Nup358/RanBP2 (na),
RanBP3 and Nup50 (Yrb2p, Nup2p)
RanGTP (iv)
Cytoplasm, NPC and nucleus,
respectively
RanBPM
(Yrb30p)
RanGTP (2H)
RanGTP (iv)
Centrosome
Cytoplasm
Function
RanGAP
RanGEF
Nuclear RanGDP import
Nuclear export
Structural components of the
NPC, nuclear import and export
Guanine-nucleotide exchange on
Ran
Intranuclear RNA processing and
degradation
Nuclear export of RanGTP and
cargo, nuclear import of cargo
GTP hydrolysis on Ran, guaninenucleotide exchange on Ran,
nuclear import and export
Mitosis
Unknown
Abbreviations: iv, in vitro binding; 2H, two-hybrid interaction; NPC, nuclear pore complex; na, not applicable.
by an importin αβ heterodimer, in vitro (Lane et al., 2000;
Paschal et al., 1996; Tachibana et al., 1996).
The recent crystal structure of the RCC1-Ran complex
(Renault et al., 2001) suggests that NTF2 and RCC1 cannot
bind simultaneously to RanGDP and, thus, RanGDP has to
dissociate from NTF2 for nucleotide exchange. Such a
conclusion is consistent with the reported guanine-nucleotidedissociation inhibitor (GDI) activity of NTF2 (Yamada et al.,
1998) and the need for nuclear RanGTP-binding proteins, in
addition to NTF2, if efficient nuclear accumulation of Ran is
to occur in a permeabilized cell system (Ribbeck et al., 1998).
These findings suggest that release and guanine-nucleotide
exchange on RanGDP in the nucleus require additional factors.
These factors would also drive the guanine-nucleotideexchange reaction in the right direction, since RCC1 has no
intrinsic preference for either of the two nucleotide-bound
states of Ran. Possible candidates for such factors are RanBP1
and Mog1p (see below).
Recently, Katahira et al. identified a protein that has
significant similarity (26% sequence identity) to NTF2, NXT1
(NTF2-related nuclear export protein 1; p15) in
coimmunoprecipitation experiments with TAP, an essential
mRNA export factor in mammalian cells (Katahira et al.,
1999). Interestingly, NXT1 forms a heterodimer with a domain
of TAP that shows similarity to NTF2 (Strässer et al., 2000;
Suyama et al., 2000). It can coactivate TAP-dependent RNA
export both in yeast and in mammalian cells (Braun et al.,
2001; Guzik et al., 2001; Katahira et al., 1999) but also
enhances a variety of other nuclear export pathways, which
suggests that it is a general nuclear export factor (Black et al.,
1999; Ossareh-Nazari et al., 2000). In accordance with such a
role, NXT1/p15 has been localized to the nucleoplasm and the
NPC at steady state and shown to shuttle between the nucleus
and the cytoplasm (Black et al., 1999). Like NTF2, NXT1
binds directly to Ran, however, in contrast to NTF2 binding,
binding of NXT1 is apparently specific for the GTP-bound
form of Ran. This interaction, although controversial because
it has been observed only in one laboratory so far, appears to
be important for the role of NXT1 in RNA export (OssarehNazari et al., 2000). The exact function of NXT1 in nuclear
export is unclear. In Crm1-dependent nuclear protein export,
NXT1 facilitates the terminal release of the export complexes
from the NPC in the cytoplasm (Black et al., 2001), which
suggests it is involved in late steps of this process.
RanGDP-binding Zn-finger nucleoporins
Besides NTF2, only two other RanGDP-binding proteins have
been identified, Nup358/RanBP2 and Nup153 (Nakielny et al.,
1999; Saitoh et al., 1996; Yaseen and Blobel, 1999b). Both
proteins are components of the NPC and are thus probably
responsible for the association of RanGDP with the NPC
(Görlich et al., 1996). Nup358/RanBP2 and Nup153 bind to
RanGDP through a common Zn-finger motif that has also been
implicated in the interaction with Crm1, a nuclear export
receptor of the importin β family, and DNA, respectively
(Singh et al., 1999; Sukegawa and Blobel, 1993). Interestingly,
the two nucleoporins are located on different sides of the NPC,
Nup358/RanBP2 being a component of the cytoplasmic
filaments and Nup153 localizing to the nuclear basket of the
NPC. The exact role of these RanGDP-binding sites on both
sides of the NPC is not clear. The implication of both
nucleoporins in nuclear import and export suggests that the
RanGDP-binding domains are involved in the fine tuning of the
Ran GTPase cycle for nucleocytoplasmic transport, although
this has not yet been addressed experimentally (Kehlenbach et
al., 1999; Shah et al., 1998; Singh et al., 1999; Yaseen and
Blobel, 1999a). Alternatively, these domains could play a role
in Ran-dependent NE formation during mitosis in higher
eukaryotes (see below). In accordance with such a hypothesis,
both Nup358/RanBP2 and Nup153 have been shown to be
among the first nucleoporins incorporated in the newly forming
NE after chromosome segregation (Bodoor et al., 1999;
Haraguchi et al., 2000). Indeed, yeasts, which do not undergo
nuclear breakdown during mitosis, do not have Zn-fingerdomain-containing nucleoporins.
The RanBP1 family of RanGTP-binding proteins
RanBP1 (Ran-binding protein 1) was the first Ran-binding
protein identified (Coutavas et al., 1993; Table 1). The protein
binds specifically to RanGTP through a highly conserved
~140-residue Ran-binding domain (RBD; also known as
RanBP1-homologous domain, RBH; Beddow et al., 1995;
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JOURNAL OF CELL SCIENCE 114 (18)
Lounsbury et al., 1994). This domain is also found in the giant
nucleoporin Nup358/RanBP2 (see above), in which several
copies are present, and in a group of nuclear proteins
represented by Nup2p and Yrb2p in S. cerevisiae. Homologues
of the latter group of proteins include Hba1p in
Shizosaccharomyces pombe and Nup50 and RanBP3 in
humans (Guan et al., 2000; Mueller et al., 1998; Smitherman
et al., 2000; Turi et al., 1996; Welch et al., 1999).
Human RanBP1 and its S. cerevisiae ortholog, Yrb1p,
shuttle rapidly between the nucleus and the cytoplasm and
localize exclusively to the cytoplasm at steady state (Künzler
et al., 2000; Plafker and Macara, 2000). Interestingly, nuclear
export of RanBP1 depends on Crm1 in both organisms,
although the export receptor might recognize different export
signals. The nuclear function of RanBP1 remains largely
obscure (see below), but the protein is thought to play an
important role in the dissociation of export complexes and the
recycling of import receptors in the cytoplasm. RanBP1 and
isolated RBDs were shown to inhibit exchange but to
coactivate hydrolysis of Ran-bound GTP in vitro (Bischoff et
al., 1995). More importantly, these domains can overcome the
inhibition of GTP hydrolysis imposed by formation of
complexes between RanGTP and members of the importin β
family of nuclear transport receptor (Bischoff and Görlich,
1997; Floer et al., 1997; Lounsbury and Macara, 1997; see
below). The dissociation of such complexes is a crucial step
both in the release of cargo from nuclear export complexes and
in the recycling of nuclear import receptors since export and
import receptors of the importin β family leave the nucleus in
complex with RanGTP (Fig. 1A; see below). Interestingly, the
level of RanBP1 in mammalian cells is cell cycle regulated,
increasing from S phase to M phase, peaking in metaphase and
abruptly declining in late telophase (Guarguaglini et al., 2000).
This cycling might reflect the role of Ran during mitosis in
higher eukaryotes (see below), given that there is no evidence
for such regulation in yeast, in which the NE does not
disassemble during mitosis (Bäumer et al., 2000).
Nup358/RanBP2 contains several functional domains,
including RanGDP-binding Zn-finger domains (see above) and
several RanGTP-binding RBDs. The latter, maybe in
conjunction with the RanGDP-binding Zn-finger domains,
presumably have the same role as the cytoplasmic RanBP1
(Kehlenbach et al., 1999). The additional function of
Nup358/RanBP2 as docking site for RanGAP1 (see above)
may further facilitate this function. In the case of the classical
nuclear import pathway, the RBDs of Nup358/RanBP2 have
been shown to link initiation and termination steps (Yaseen and
Blobel, 1999a). As in the case of Zn-finger domains, the RBDs
might play an additional role in nuclear envelope formation
during mitosis. The structure of a co-crystal containing a RBD
of RanBP2 and RanGppNHp revealed a unique molecular
embracement between the N-terminal domain of the RBD and
the C-terminal domain of Ran (Vetter et al., 1999a). The core
domain of the RBD that makes direct contacts with the switch
I region of Ran exhibits the same topology as pleckstrin
homology (PH) domains implicated in the binding of
phosphoinositide lipids, which indicates that this fold
represents a versatile interaction module.
The RBDs of nuclear RanBP1-family proteins, exemplified
by S. cerevisiae Nup2p and Yrb2p, are rather degenerate, and
their affinities for RanGTP are low – in fact, Mueller et al.
proposed that is a prerequisite for their function in the nucleus
(Mueller et al., 1998). However, there is recent evidence that
these proteins, although nuclear at steady state, shuttle between
the nucleus and cytoplasm (Dilworth et al., 2001; Lindsay et
al., 2001). Both Nup2p and Yrb2p contain FG repeats, as do
some of the nucleoporins. These repeats have an affinity for
various nuclear transport receptors. Nup2p and Yrb2p have
been implicated in Cse1p- and Crm1p-dependent export,
respectively, although their exact role in these processes is
unclear (Booth et al., 1999; Hood et al., 2000; Solsbacher et
al., 2000; Taura et al., 1998). Identifying a possible
mechanism, Lindsay et al. have recently shown that RanBP3,
the mammalian homologue of Yrb2p, enhances Crm1mediated nuclear export by increasing the affinity of the
receptor for its cargo (Lindsay et al., 2001).
Karyopherins
The family of importin-β-like nuclear transport receptors
(karyopherins) comprises 14 different members in S. cerevisiae
(Adam, 1999). The number of mammalian proteins known to
belong to this family is steadily increasing. Members of this
family have a repetitive structure that accommodates binding
to RanGTP, cargo and the NPC (Mattaj and Conti, 1999). They
contain a degenerate ~150-residue RanGTP-binding motif at
the N-terminus, which shows no similarity to the RBD of
RanBP1. Crystal structures of two importins reveal an archlike overall structure in which the inner face of the arch binds
to RanGTP or cargo in a mutually exclusive manner, and the
outer face of the arch makes contacts with FG-repeat sequences
in nucleoporins (Bayliss et al., 2000; Chook et al., 1999).
Binding sites for importin β and the RBD on RanGTP do not
overlap, which is consistent with the formation of trimeric
complexes involving these proteins in vitro (Chi et al., 1997;
Vetter et al., 1999b). Such complexes are possible
intermediates in the dissociation of karyopherin-RanGTP
complexes (see above). Binding of RanGTP to karyopherins
thereby not only inhibits RCC1-mediated guanine-nucleotide
exchange and RanGAP1-mediated GTP hydrolysis on Ran but
also modulates the interaction of karyopherins with cargo and
the NPC. In the case of importins, binding of RanGTP releases
cargo and the NPC, whereas in the case of exportins binding
is required for their association with cargo and the NPC. It will
be interesting to see how such similar proteins can exhibit such
a different behaviour.
Mog1 and Dis3
Oki and Nishimoto identified Mog1p (multicopy suppressor of
gsp1) in a genetic screen for multicopy suppressors of
temperature-sensitive mutations in GSP1, which encodes yeast
Ran (Oki and Nishimoto, 1998). Mog1p is a nuclear protein
that is evolutionarily conserved (Marfatia et al., 2001;
Tatebayashi et al., 2001). It binds to both nucleotide-bound
forms of Ran and competes with NTF2 for binding to RanGDP
(Stewart and Baker, 2000). The crystal structure reveals a
unique fold, although the topology of some elements resembles
part of NTF2 (Stewart and Baker, 2000). mog1-null S.
cerevisiae mutants exhibit a temperature-sensitive phenotype
that is suppressed by high-copy numbers of NTF2 and GSP1
(Oki and Nishimoto, 1998). The mutant exhibits defects in
nuclear protein import but not in the nuclear export of
poly(A)+-RNA (Oki and Nishimoto, 1998). In S. pombe,
Targeting of Ran: variation on a common theme?
Mog1p is essential for viability, and a temperature-sensitive
mutation leads to defects in nuclear protein import and nuclear
accumulation of poly(A)+-RNA (Tatebayashi et al., 2001).
Overexpression of S. pombe Ran, Spi1p, rescues S. pombe
mog1∆ cells from death. Remarkably, the S. pombe knockout
is complemented by Xenopus laevis and S. cerevisiae Mog1p,
which suggests that the function of Mog1p is conserved from
yeast to frog (Nicolás et al., 2001).
At present, the role of Mog1 in the Ran GTPase cycle is
unclear. The genetic interaction with NTF2 suggests that Mog1
plays a role in the nuclear accumulation of Ran. Indeed, Gsp1p
is already relocalized to the cytoplasm in a mog1∆ strain at the
permissive temperature (Stochaj et al., 2000), which is similar
to the situation in prp20-1 cells (Fig. 1B). Thus, an increase in
the cellular level of Ran is able to rescue three different
mutations in S. cerevisiae (prp20-1, ntf2 and mog1∆) that are
all defective in nuclear accumulation of Gsp1p. These findings,
together with the fact that Mog1 is located in the nucleus and
binds to Ran irrespectively of the nucleotide-bound state (and
also binds to the nucleotide-free state), suggest that the protein
plays a role in the release of RanGDP from NTF2 and in the
subsequent GDP-to-GTP exchange on Ran by RCC1. In
accordance with such a hypothesis, two groups recently
demonstrated that Mog1 acts as a guanine-nucleotide-release
factor for Ran in vitro (Oki and Nishimoto, 2000; Steggerda
and Paschal, 2000). However, this activity was on GTP-to-GDP
exchange and not the physiological inverse reaction. In fact,
Mog1 alone inhibits the latter reaction. Interestingly, most
recent results suggest that RanBP1 can relieve this inhibition
and contribute to cooperative coactivation of RCC1-mediated
GDP-to-GTP exchange on Ran, which is similar to the role of
RanBP1 in RanGAP1-mediated GTP hydrolysis (Nicolás et al.,
2001). In accordance with such a model, mutations in YRB1
and PRP20 are conditionally lethal with a deletion of MOG1
(M.K. and E.H., unpublished). This model would also explain
the nucleocytoplasmic shuttling of RanBP1.
Dis3p has Ran-binding characteristics similar to those of
Mog1p (Noguchi et al., 1996; Shiomi et al., 1998). It was
identified in a two-hybrid screen using human Ran as a bait
and is conserved from yeast to man. In vitro, Dis3p binds to
both nucleotide-bound forms of Ran but also to the nucleotidefree form of Ran. In addition, it can enhance the RanGEF
activity of RCC1 and is found in complex with Ran and RCC1
in vivo (Noguchi et al., 1996). Recently, Mitchell et al. reported
that Dis3p is identical to Rrp44p, a component of a nucleolar
RNA-processing complex called the exosome (Mitchell et al.,
1997). Dis3p/Rrp44p itself has 3′-5′ exoribonuclease activity,
but it is unclear how this activity relates to its in vitro activity
as a coactivator of RCC1-mediated guanine-nucleotide
exchange on Ran. Suzuki et al. recently proposed that Ran
regulates exosome assembly/disassembly through Dis3p
independently of its role in nucleocytoplasmic transport
(Suzuki et al., 2001). In any case, mutations in the essential S.
cerevisiae DIS3 gene lead to nuclear accumulation of various
RNAs, which might be a consequence of defects in their
processing (Gadal et al., 2001; Grosshans et al., 2001).
RanBPM and Yrb30p
Nakamura et al. isolated RanBPM in a two-hybrid screen using
Ran as a bait (Nakamura et al., 1998). They showed that
RanBPM binds specifically to the GTP-bound form of Ran in
3237
the two-hybrid system. To date, no direct binding of RanBPM
to Ran in vitro has been demonstrated. Interestingly, the protein
localizes to centrosomes and its overexpression induces ectopic
nucleation of microtubules. This phenotype, in conjunction
with the two-hybrid interaction with Ran, suggests that
RanBPM is a target of Ran in microtubule aster formation (see
below). RanBPM displays considerable similarity to the Cterminal half of an S. cerevisiae ORF, but the relevance of this
similarity remains to be shown (Nakamura et al., 1998).
Yrb30p is an S. cerevisiae ORF that was identified in a twohybrid screen using the hydrolysis-defective Gsp1pG21V
mutant as a bait (A. Braunwarth, M. Fromont-Racine, P.
Legrain, R. Bischoff, E.H. and M.K., unpublished). The protein
interacts specifically with the GTP-bound form of Gsp1p in
vitro. Interestingly, Yrb30p does not show any sequence
similarity to either RanBP1 or importin β and appears to be
localized exclusively to the cytoplasm. The cellular function of
this novel RanGTP-binding protein is unknown.
The multiple functions of Ran
Nucleocytoplasmic transport
Since the identification of Ran as a protein necessary for
nuclear protein import (Moore and Blobel, 1993), the field of
nucleocytoplasmic transport has boomed. As a result, today we
have a relatively precise understanding of the mechanism of
this essential cellular process (Görlich and Kutay, 1999;
Nakielny and Dreyfuss, 1999; see animation in; Allen et al.,
2000). Ran plays an eminent role in this process, helping to
establish compartmental identitity and providing the
directionality of transport (Macara et al., 2000). In contrast to
early hypotheses, the current view is that GTP hydrolysis on
Ran is not required as an energy source for the translocation
process. There is an asymmetric distribution of RanGTP (and
RanGDP) across the nuclear envelope (see above), and
directionality of transport is provided by the different
modulation of importins and exportins by RanGTP with
respect to their cargo binding. Whereas importins bind to
RanGTP and cargo in a mutually exclusive manner, exportins
bind to RanGTP and cargo cooperatively. As a consequence,
formation of importin-cargo and exportin-cargo-RanGTP
complexes is restricted to the cytoplasm and the nucleoplasm,
respectively. Indeed, inversion of the RanGTP gradient in vitro
leads to an inversion of the directionality of nucleocytoplasmic
transport (Nachury and Weis, 1999). At this point, we would
like to emphasize that, among all Ran-binding proteins
discussed above, the family of importin-β-like nuclear import
and export receptors represents the only direct link between the
Ran GTPase cycle and a cellular process. In this strict sense,
members of this family of proteins represent the only real Ran
targets known so far.
Spindle formation
The identification of a centrosomal protein, RanBPM, as a
Ran-binding protein that induces ectopic microtubule
nucleation when overexpressed suggested a possible role for
Ran in chromosome segregation (see above). Studies using
mitotic extracts from X. laevis eggs have shown that Ran is
necessary for centriole-dependent microtubule aster formation,
can stabilize microtubule asters and even promote spindle
formation when it is exogenously added (Kahana and
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JOURNAL OF CELL SCIENCE 114 (18)
Cleveland, 1999). Interestingly, this activity of Ran, which
occurs in the absence of nuclei and is thus independent of its
role in nucleocytoplasmic transport, relies on the GTP-bound
form of the GTPase. GTP hydrolysis does not seem to be
important, because mutant forms of RanGTP that cannot
hydrolyze GTP can do the job. These activities, however,
require other proteins in the egg extract: RanGTP cannot
stimulate microtubule polymerization from purified tubulin
subunits. Such proteins include motor proteins (dynein) and
other centrosome-associated factors (NuMA, TPX2,
XGRIP109 and XMAP215).
The mechanism of this additional function of Ran had
remained obscure until recently, but three groups have now
reported that, in these extracts, RanGTP acts by releasing
microtubule-associated proteins (MAPs) TPX2 and NuMA
from importin β (Gruss et al., 2001; Nachury et al., 2001;
Wiese et al., 2001). This suggests that the role of RanGTP in
microtubule nucleation is very similar to its role in
nucleocytoplasmic transport. In both cases, high local
concentrations of RanGTP, either close to chromatin (due to
chromatin-bound RCC1) or within the nucleus, release import
cargo from their receptor. During mitosis, the MAPs released
in such a way are able to trigger microtubule nucleation owing
to the absence of an NE. During interphase, these proteins are
sequestered away from the cytoplasmic tubulin into the nucleus
possibly in order to prevent premature microtubule nucleation.
It has been speculated that RanGTP-induced spindle formation
is important in plants and in some meiotic cells that lack a
defined microtubule-organizing center like a centriole.
However, there is evidence that Ran and importin β perform
mitotic functions even in somatic cells (Guarguaglini et al.,
2000; Nachury et al., 2001), which argues for a more general
mechanism.
Nuclear envelope formation
Besides its role in nucleocytoplasmic transport and spindle
formation, Ran is also required for NE assembly in chromatinfree Xenopus egg extracts (Hetzer et al., 2000; Zhang and
Clarke, 2000) and human somatic cell extracts (Zhang and
Clarke, 2001). In contrast to microtubule nucleation, NE
assembly requires both GTP hydrolysis and guaninenucleotide exchange on Ran, since non-hydrolyzable GTP
analogues and mutants of Ran incapable of GTP hydrolysis
(RanQ69L) or nucleotide-binding (RanT24N) inhibit this
process. In addition, depletion of RCC1 and RanGAP1 from
the extracts abolishes NE assembly. The Ran-induced NE is
continuous and contains NPCs that support active nuclear
import and export. The molecular mechanism involved is
currently not known, because no targets of Ran in this process
have been identified to date. The RanGDP- and/or RanGTPbinding nucleoporins are candidates for such proteins (see
above). A high local concentration of RanGTP on chromatin
appears to promote recruitment of membrane vesicles, and a
localized Ran GTPase cycle accomplished by RCC1 and
RanGAP1 on chromatin then leads to vesicle fusion.
Conclusions
It is clear now that Ran functions in at least three different
processes: nucleocytoplasmic transport, microtubule aster
formation and NE formation. Although the molecular details
of its roles in latter two processes are still obscure, all appear
to depend on the localized generation of RanGTP by
chromatin-bound RCC1. Remarkably, the latest reports now
indicate that all these functions could rely on the same class of
target protein, karyopherins. Intriguingly, these proteins
represent, in a strict sense, the only direct Ran-targets known
so far. Weis and co-workers have speculated that the function
of these proteins in microtubule aster and NE formation
preceded their function in nucleocytoplasmic transport
(Nachury et al., 2001). Alternatively, the additional functions
of Ran during open mitosis in higher eukaryotes might have
evolved from the situation in fungi, in which proteins involved
in spindle formation have to be imported into the nucleus
because the NE does not disassemble during mitosis (Wiese et
al., 2001).
It will be interesting to see whether importin β also plays a
role in NE formation and whether there are Ran targets in
microtubule aster formation and NE formation, besides
importin β, that are specific for these cellular processes. Finally,
it would be interesting to know whether these additional roles
of Ran are restricted to eukaryotic organisms that exhibit open
mitosis or whether there are similar roles in fungi that do not
undergo nuclear breakdown during mitosis. In metazoans at
least, Ran can be regarded as a marker for the nuclear
compartment during interphase, as a marker for condensed
chromatin during metaphase and anaphase and as a marker for
decondensed chromatin during telophase (Hetzer et al., 2000).
We thank all members of the Hurt laboratory for helpful discussions
and Helge Grosshans, George Simos and Ralf Bischoff for critical
reading of the manuscript.
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