Molecular Cell
Article
Arx1 Functions as an Unorthodox Nuclear Export
Receptor for the 60S Preribosomal Subunit
Bettina Bradatsch,1 Jun Katahira,2 Eva Kowalinski,1 Gert Bange,1 Wei Yao,1 Toshihiro Sekimoto,2
Viola Baumgärtel,3 Guido Boese,3 Jochen Bassler,1 Klemens Wild,1 Reiner Peters,3 Yoshihiro Yoneda,2
Irmi Sinning,1 and Ed Hurt1,*
1Biochemie-Zentrum
der Universität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
for Biomolecular Networks, Department of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka
565-0871, Japan
3Medical Physics and Biophysics, Center of Nanotechnology, Gievenbecker Weg 11, D-48149 Münster, Germany
*Correspondence: [email protected]
DOI 10.1016/j.molcel.2007.06.034
2Laboratories
SUMMARY
Shuttling transport receptors carry cargo
through nuclear pore complexes (NPCs) via
transient interactions with Phe-Gly (FG)-rich nucleoporins. Here, we identify Arx1, a factor associated with a late 60S preribosomal particle
in the nucleus, as an unconventional export receptor. Arx1 binds directly to FG nucleoporins
and exhibits facilitated translocation through
NPCs. Moreover, Arx1 functionally overlaps
with the other 60S export receptors, Xpo1 and
Mex67-Mtr2, and is genetically linked to nucleoporins. Unexpectedly, Arx1 is structurally unrelated to known shuttling transport receptors
but homologous to methionine aminopeptidases (MetAPs), however, without enzymatic
activity. Typically, the MetAP fold creates a central cavity that binds the methionine. In contrast, the predicted central cavity of Arx1 is involved in the interaction with FG repeat
nucleoporins and 60S subunit export. Thus, an
ancient enzyme fold has been adopted by
Arx1 to function as a nuclear export receptor.
INTRODUCTION
Ribosome biogenesis in eukaryotic cells takes place sequentially in the nucleolar, nucleoplasmic, and cytoplasmic compartments and consists of many steps that are
highly interconnected and tightly controlled (Warner,
1999). Insight into the spatio-temporal events during preribosome subunit formation initially came from pulse/
chase and sucrose gradient analysis and more recently
from purification of various preribosomal particles via the
tandem-affinity-purification (TAP) method (Tschochner
and Hurt, 2003). These analyses revealed that numerous
nonribosomal factors are transiently associated with nascent ribosomes. Moreover, proteomics and intracellular
localization studies of the isolated preribosomes allowed
grouping of the large number of nonribosomal factors associated with pre-90S, pre-60S, and pre-40S particles
into different classes along the maturation pathway (Fatica
and Tollervey, 2002; Tschochner and Hurt, 2003). Electron
microscopy has revealed the structure of preribosomal
particles, including pre-90S particles and a tadpole-like
pre-60S particle that differs from the mature 60S subunit
(Dragon et al., 2002; Nissan et al., 2004).
The earliest preribosomal particle that is formed during
pre-rRNA transcription consists predominantly of small
subunit proteins, the U3 snoRNP, and 35 nonribosomal
factors (Dragon et al., 2002; Grandi et al., 2002). The transition from pre-90S to pre-40S particles is initiated by
cleavage of the nascent pre-rRNA accompanied by removal of the bulk of ‘‘90S biogenesis factors.’’ The resulting 40S preribosome contains only a handful of nonribosomal proteins (Schäfer et al., 2003). At this transition, the
earliest pre-60S particles, which are assembled around
the 27S pre-rRNA and exhibit a more complicated composition than the pre-40S particles, can be detected. Both
pre-40S and pre-60S particles then follow separate maturation routes through the nucleolus and nucleoplasm before export to the cytoplasm (Johnson et al., 2002).
During 60S subunit biogenesis, the composition of the
pre-60S particles changes significantly. Early pre-60S
particles contain a large number of nonribosomal factors
(Baßler et al., 2001; Harnpicharnchai et al., 2001; Saveanu
et al., 2001; Fatica et al., 2002). During maturation, which
proceeds in the nucleolus and nucleoplasm, many of the
factors leave the particle, while only few additional factors
associate (Nissan et al., 2002). Before exit to the cytoplasm, export factors join the late nuclear pre-60S particle
(Nissan et al., 2002). Among these factors is the conserved
adaptor protein Nmd3, which contains a nuclear export
sequence (NES) that is recognized by the nuclear export
receptor Crm1/Xpo1 (Ho et al., 2000; Gadal et al., 2001;
Thomas and Kutay, 2003; Trotta et al., 2003). Surprisingly,
the export receptor for mRNA, Mex67-Mtr2 (Santos-Rosa
et al., 1998), also plays a role in the nuclear export of the
60S subunit (Baßler et al., 2001). Recent findings showed
that the Mex67-Mtr2 heterodimer is recruited to the late
pre-60S subunit via a distinct interaction surface on the
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Arx1 Is a Nuclear Export Receptor for 60S Subunits
receptor that does not participate in mRNA export (Yao
et al., 2007).
After export to the cytoplasm, the Nmd3 adaptor is released from the 60S ribosomal subunit, which was suggested to require its exchange with the ribosomal Rpl10
protein and regulation by the cytoplasmic GTPase Lsg1
(Hedges et al., 2005; West et al., 2005). Recently, additional factors associated with preribosomal particles
were implicated in nuclear export. One example is
Rrp12, which was proposed to facilitate export of both ribosomal subunits via its interactions with nucleoporins
and Ran (Oeffinger et al., 2004).
Here, we describe Arx1 as an unusual nuclear export
factor of the pre-60S subunit. Arx1, a nucleo-cytoplasmic
shuttling protein (Belaya et al., 2006), is associated with
a late nucleoplasmic pre-60S particle that contains the
Nmd3 export adaptor and the Mex67-Mtr2 exporter (Nissan et al., 2002; Yao et al., 2007). Arx1 can directly bind to
a subset of FG nucleoporins and translocate through the
NPCs. Moreover, in vivo Arx1 is genetically linked to
Nmd3 and Mex67-Mtr2. However, Arx1 is not homologous to known shuttling transport receptors but exhibits
a methionine aminopeptidase fold. This enzyme fold is utilized by Arx1 to bind to both the pre-60S subunit and FG
repeat nucleoporins, a process that, in turn, facilitates nuclear export of the pre-60S subunit.
RESULTS
Arx1 Interacts Genetically with Pre-60S Export
Factors and Nuclear Pore Proteins
Arx1 is associated with a late pre-60S particle that carries
nuclear export factors such as Nmd3 and Mtr2 (Nissan
et al., 2002). This finding prompted us to test whether
Arx1 has a role in pre-60S subunit export. However,
ARX1 is not essential for cell growth, and an arx1 disruption strain (arx1D) does not have a growth defect at 30 C
(Figure 1A). Thus, we tested whether Arx1 interacts genetically with factors involved in pre-60S export. Indeed, the
arx1D strain exhibits synthetic lethal (sl) or synthetic enhanced (se) growth defects when combined with mutations that map in known pre-60S subunit export factors
(e.g., nmd3DNES1, xpo1-1, mtr2-33, and ecm1D)
(Figure 1A). Previously, mex67 mutant alleles were isolated that specifically impaired 60S subunit, but not
mRNA export (Yao et al., 2007). These mex67 alleles
(mex67Dloop, mex67loop KR>AA) were also se or sl
when combined with the arx1D strain (Figure 1A). On the
other hand, mutant alleles of pre-60S biogenesis factors
(e.g., nug1-2, rea1-7, rea1-21, rix1-1, yph1-1, yph1-2,
and cic1-2) did not genetically interact with ARX1
(Figure 1A and data not shown). Moreover, the function
of the pre-60S factor Alb1 that forms a stable heterodimeric complex with Arx1 (Lebreton et al., 2006; see also
Figure S1A in the Supplemental Data available with this article online) is genetically unlinked to the Arx1 function
(Figure S1B). Taken together, these genetic studies indicated that Arx1 functionally interacts with export, but not
biogenesis, factors of the 60S subunit. However, Arx1 is
not involved in the recruitment of these export factors, because Nmd3 and Mex67-Mtr2 were still bound to the pre60S subunit in a yeast strain lacking ARX1 (Figure S1C). In
contrast, Arx1 targets Alb1 to the pre-60S particle, but
binding of Arx1 to pre-60S is not dependent on Alb1
(Figure S1D).
To identify additional proteins that functionally interact
with ARX1, we performed a synthetic lethal screen with
the arx1D allele. Two synthetic lethal mutants were isolated that were complemented by ECM1 (see also
Figure 1A) and GLE2, respectively (Figure 1B). Gle2 is
a yeast nuclear pore protein that physically associates
with the GLFG repeat nucleoporin Nup116 (Bailer et al.,
1998) and is involved in poly(A)+ RNA export (Murphy
et al., 1996; Bailer et al., 1998). The direct combination
of the arx1D and gle2D disruption alleles caused a strong
se phenotype when compared to the single mutants
(Figure 1C). Moreover, arx1D/nup116D and arx1D/nup42D
strains showed a synthetically enhanced growth defect
(Figures 1D and 1E), whereas the combination of arx1D
with nup100D did not induce such a phenotype (data
not shown). Thus, the Arx1 function also depends on
components of the nuclear pore complex.
A Role of Arx1 in Nuclear Export of 60S Subunits
To show that Arx1 plays a role in the transport of the 60S
subunit to the cytoplasm, we analyzed the nuclear export
of Rpl25-GFP (a large subunit reporter) and Rps2-GFP
(a small subunit reporter) in the synthetically enhanced
arx1D mutants. The single mutants arx1D, gle2D,
ecm1D, and mex67Dloop did not show an apparent 60S
subunit export defect. However, the double mutants
arx1D/gle2D, arx1D/ecm1D, and arx1D/mex67Dloop accumulated the Rpl25-GFP reporter in the nucleus, suggesting that 60S subunit export was inhibited
(Figure 2A). In contrast, nuclear export of the 40S subunit
was not affected in these mutants (Figure 2A). Moreover,
the arx1D/mex67Dloop strain defective in ribosome export was not impaired in mRNA export (Figure 2B).
The nup116 disrupted strain (nup116D) already exhibited a mild 60S export defect, which can be seen as
concentration of the Rpl25-GFP reporter around the nuclear envelope in a few cells (Figure 2A). However, in the
arx1D/nup116D double mutant, Rpl25-GFP accumulated
more strongly in the nucleus and at the nuclear periphery
in an increased number of cells (Figure 2A). Thus, when
cells lack ARX1, a 60S subunit export defect is induced
by mutations in nucleoporins or pre-60S export factors.
Arx1 Binds Directly to FG Repeat Domains
of Nucleoporins
The data so far obtained suggested that Arx1 functions as
a 60S export factor. Interestingly, Ydr101c (Arx1) was detected in FG repeat nucleoporin pull-downs (Allen et al.,
2001). To investigate whether recombinant Arx1 can
bind directly to FG repeat nucleoporins, purified Arx1
was incubated with different FG repeat nucleoporins in
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Arx1 Is a Nuclear Export Receptor for 60S Subunits
Figure 1. ARX1 Is Genetically Linked to Components of the 60S Subunit Export Machinery and the Nuclear Pore Complex
(A) Synthetic lethality (sl) or synthetic enhancement (se) of growth retardation of the arx1D strain combined with the indicated mutant alleles
nmd3DNES1, xpo1-1, mtr2-33, mex67Dloop, mex67loop KR>AA, and ecm1D. The temperature-sensitive alleles mtr2-21, nug1-2, rea1-7, and
rea1-21 served as negative controls. The strains carrying the indicated wild-type and mutant alleles were spotted in serial 10-fold dilutions onto
SDC (when se) or SDC/5-FOA plates (when sl) and incubated at 30 C for 3–5 days.
(B) A synthetic lethal screen identifies ECM1 and nucleoporin GLE2 to genetically interact with ARX1. Two sl mutants were isolated that exhibited
a nonsectoring red phenotype and no growth on SDC/5-FOA plates. Sl102 was complemented by ECM1 and sl101 by GLE2. Cells were grown
on SDC and SDC/5-FOA plates at 30 C for 3 or 5 days.
(C–E) Synthetic enhanced phenotypes of arx1D/gle2D (C), arx1D/nup116D (D), and arx1D/nup42D (E) double mutants. Cells expressing wild-type
alleles from plasmid were spotted in serial 10-fold dilutions and incubated at 30 C for 3 or 2 days (E).
the presence of an E. coli cell lysate to compete for unspecific binding (Figure 3A). As a positive control, we performed the binding assay in parallel with recombinant purified Mex67-Mtr2, an export receptor that is known to
interact with several FG nucleoporins (Figure 3A; Sträßer
et al., 2000). Arx1 was found to bind efficiently to GSTNup100, GST-Nup42, and GST-Nup116 but only weakly
to GST-Nup1 repeats. However, Arx1 did not bind to the
C-terminal coiled-coil domain of Nsp1, which lacks the
FG repeats (Figure 3A). Unexpectedly, the human homo-
log of Arx1 called Ebp1 did not associate with FG repeat
nucleoporins (Figure S2A). Moreover, yeast methionine
aminopeptidase (Map1), which shows homology to Arx1
and Ebp1 (see below), could not bind to FG repeat nucleoporins (Figure S2B).
To obtain quantitative data on the binding of Arx1 to FG
nucleoporins, BIAcore measurements were performed.
The GLFG repeat domain of Nup100 (good binder) and
the FXFG repeat domain of Nup1 (weak binder) were immobilized on a sensor chip, and varying concentrations
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Arx1 Is a Nuclear Export Receptor for 60S Subunits
Figure 2. Nuclear Export of 60S Subunits
Is Impaired in arx1D/gle2D, arx1D/
ecm1D, arx1D/nup116D, and arx1D/
mex67Dloop Mutants
(A) Analysis of nuclear export of 60S and 40S
subunits in synergistic enhanced arx1D mutants. The indicated single and double mutant
strains expressing either the 60S subunit reporter Rpl25-GFP or 40S subunit reporter
Rps2-GFP were grown in SDC medium at
23 C (arx1D/gle2D and arx1D/ecm1D) or
30 C (arx1D/nup116D, rio2-1, and arx1D/
mex67Dloop). Subcellular location of Rpl25GFP and Rps2-GFP was analyzed by fluorescence microscopy. The rio2-1 mutant served
as positive control for nuclear accumulation
of Rps2-GFP.
(B) arx1D does not cause a poly(A)+ RNA export defect when combined with mex67 mutant
alleles. The arx1D/mex67D strain expressing
the corresponding alleles was grown in SDC
at 23 C and shifted to 37 C for 1 hr to analyze
poly(A)+ RNA export. DNA was stained with
DAPI. mex67-5 served as positive control for
nuclear poly(A)+ RNA accumulation.
of purified Arx1 were applied. Arx1 showed a clear but
transient interaction with the FG repeat domain of
Nup100 (Figure 3B). Accordingly, the sensorgram exhibited a typical rectangular shape indicative of a high dissociation rate. Scatchard analysis revealed the apparent
dissociation constant (Kd) of 1.84 mM for the binding between Arx1 and GST-Nup100 (Figure 3B). The interaction
between Arx1 and Nup1 FG repeats was too weak to calculate a reliable Kd value (Figure 3B). Thus, Arx1 binds to
GLFG repeats with a Kd in the low mM range.
Arx1 Exhibits Facilitated Translocation through
the Nuclear Pore Complex
The observed interaction of Arx1 with FG nucleoporins
prompted us to analyze whether fluorescently labeled
Arx1 (Figure 4A) can directly pass through the nuclear
pore complexes using an in vitro NPC translocation assay
(Adam et al., 1990; Imamoto et al., 1995). In the presence
of cytosol and ATP, permeabilized HeLa cells can effi-
ciently import Snail (Yamasaki et al., 2005), a nuclear localization sequence (NLS) containing cargo protein
(Figure 4B). In the absence of cytosol and ATP, nuclear accumulation of Snail was inhibited, but transport receptors
could still pass through the nuclear pore complexes (see
below). As shown in Figure 4B, fluorescently (Alexa 488)
labeled Arx1 alone or Arx1 fused to maltose binding protein (MBP) was efficiently translocated through the NPCs
in the permeabilized HeLa cells in the absence of cytosol
and ATP. To find out whether Arx1 can re-equilibrate
across the nuclear envelope after nuclear accumulation,
we further incubated the permeabilized cells for 0, 5,
and 15 min in buffer lacking Alexa 488-labeled Arx1. After
prolonged incubation in washing buffer, the nuclear signal
of Arx1 was significantly reduced, suggesting that Arx1
was re-exported. Under these conditions, a nuclear rim
staining of Arx1 was also noticed, which could correspond
to Arx1 bound to FG nucleoporins (Figure S3). The remaining nuclear staining of Arx1 after washing could be due to
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Arx1 Is a Nuclear Export Receptor for 60S Subunits
Figure 3. Arx1 Interacts Directly with FG Repeat Nucleoporins
(A) Binding of recombinant Arx1 (left panel) and Mex67-Mtr2 complex (right panel) to immobilized GST-FG repeat nucleoporin domains (GST-Nup) in
the presence of competitor E. coli lysate. Lane 1, Arx1 or Mex67-Mtr2; lane 2, Arx1 or Mex67-Mtr2 mixed with E. coli lysate (input); and lanes 3–14,
proteins bound to the indicated GST-Nups in the presence (+) or absence ( ) of Arx1 or Mex67-Mtr2. Bound proteins were eluted and analyzed by
SDS-PAGE and Coomassie staining (upper panel) or western blotting (lower panel) with anti-PentaHis (Arx1), anti-Mex67, and anti-Mtr2 antibodies.
GST-Nup position is indicated by an asterisk (*), bound Arx1 or Mex67-Mtr2 by a circle. S, protein standard.
(B) BIAcore analysis of the interaction between Arx1 and the FG repeat domain of Nup100. GST-Nup100 (left panel) and GST-Nup1 (middle panel)
were immobilized on sensor chips. Different concentrations of recombinant purified Arx1 were then injected over the sensor chips. Arx1-GST-Nup100
binding was analyzed by Scatchard analysis (right panel). Sensor responses at equlibrium (Req) were determined for each protein concentration from
the sensorgram, and Req/concentration of Arx1 values was plotted as a function of Req.
retention of Arx1 by nuclear structures (e.g., ribosomal
particles, etc.).
Ebp1 or Map1, however, was not able to pass through
the nuclear pore complexes and remained largely excluded from the nucleus (Figure 4B), consistent with the
observation that these proteins did not bind to FG repeats in vitro (see above). Moreover, nuclear transport
of Arx1 in permeabilized HeLa cells was inhibited by
wheat germ agglutinin (WGA; Figure 4C) but still occurred
at lower temperatures (Figure 4D). Taken together, these
data suggest that Arx1 can pass through the nuclear
pore complexes via direct contact to FG repeat nucleoporins.
To demonstrate that Arx1 can also exit the nucleus, optical single transporter recording (OSTR) measurements
were performed with nuclear envelopes of Xenopus oocytes attached to arrays of microwells (called test compartments [TCs]; Peters, 2003). Fluorescently labeled
Arx1 was added to the nuclear side of the nuclear envelope. Subsequently, the appearance of Arx1 in the TCs
was monitored by confocal microscopy. Alexa 488labeled Arx1 was efficiently translocated through the nuclear pore complexes by a facilitated mechanism at
a rate constant of 4.2 ± 1.0 3 10 3 s 1 (Figures 5A and
5B and Table S3). In contrast, Texas Red-labeled dextran
of 70 kDa (dextran70) or MBP (40 kDa), which served
as negative controls, was not significantly exported
(Figure 5A and data not shown). Moreover, no significant
nuclear export of Ebp1 and methionine aminopeptidase
Map1 was observed when employing the OSTR assay
(Figure 5B and Table S3).
When export of Arx1 into the TCs was completed, reimport was measured by removing Arx1 from the OSTR
chamber and monitoring disappearance of Arx1 from the
TCs by confocal scanning. The import rate of Arx1 was determined to be 4.5 ± 1.2 3 10 3 s 1 and therefore
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Figure 4. In Vitro Nuclear Import of Arx1 in Permeabilized HeLa Cells
(A) Purification of Arx1, Ebp1, and Map1, all tagged with His6-TEV, from E. coli BL21 cells. The proteins were labeled with Alexa 488 and analyzed by
SDS-PAGE and Coomassie staining.
(B) In vitro import assay. HeLa cells on coverslips were permeabilized by digitonin and incubated at 30 C with transport buffer containing the indicated
labeled proteins. After 5 min incubation, the cells were washed, fixed, and observed by fluorescence microscopy. The signals of GST-Snail-GFP,
Arx1, MBP-Arx1, Ebp1, and Map1 (upper panel), Alexa 546-MBP (middle panel), and phase contrast images (lower panel) are shown.
(C) Nuclear import of Arx1 is inhibited by WGA. Permeabilized HeLa cells were pretreated with transport buffer (upper panel) or transport buffer containing WGA (0.5 mg/ml; lower panel) for 15 min on ice. Then each import substrate in reaction buffer (15 mM protein, 2% BSA in transport buffer) with
or without WGA (0.5 mg/ml) was added and incubated further at 25 C for 3 min. After washing, the cells were fixed by formaldehyde and labeled
proteins were detected by fluorescence microscopy. GST-Snail-GFP was incubated with cytosol and ATP regenerating system at 25 C for 20 min.
(D) Low temperature does not inhibit the nuclear accumulation of Arx1. Nuclear import of Arx1 and Tap-p15 was tested in the permeabilized HeLa
cell system. Reactions were incubated for 15 min on ice. Alexa 488-labeled Arx1 or Tap-p15 (upper panel) and Alexa 546-MBP (lower panel) are
visualized.
comparable to the export rate. Thus, export and import
rates of Arx1 resemble each other and suggest that Arx1
can shuttle through the NPC by facilitated diffusion.
Arx1 Has a Methionine Aminopeptidase Fold but
Lacks Enzymatic Activity
Our findings indicated that Arx1 could function as an export receptor for the 60S subunit by direct interaction
with FG nucleoporins. However, Arx1 and its human homolog Ebp1 (Xia et al., 2001; Squatrito et al., 2004) exhibit
homology to MetAPs (E.C. 3.4.11.18), which catalyze the
removal of N-terminal methionine from newly synthesized
polypeptides. These enzymes contain a deep central cav-
ity inside the ‘‘pita bread’’ fold that consists of a hydrophobic substrate binding pocket and an adjacent active site,
in which hydrolytic cleavage of the peptide bond occurs
(Lowther and Matthews, 2000).
To find out whether Arx1 has methionine aminopeptidase activity, we performed enzymatic measurements
in vitro. However, recombinant and purified Arx1 did not
exhibit MetAP activity (Figure S4). In contrast, recombinant yeast methionine aminopeptidase (Map1) readily
cleaved off the N-terminal methionine from a substrate
peptide in the in vitro assay (Figure S4).
To find out if Arx1 and Ebp1 have a methionine aminopeptidase fold, we sought to crystallize these proteins.
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Figure 5. In Vitro Export of Arx1 through the Nuclear Envelope of Xenopus Oocytes
(A) OSTR analysis of nuclear export of Arx1 and dextran70. Arx1 shows facilitated diffusion into microcavities sealed by nuclear envelope (upper left
panel), whereas the control substrate dextran70 is excluded (lower left panel). Both substrates show fast diffusion into not-covered reference cavities
(upper and lower right panels, respectively). Note that the relevant signal is located in the center of each well.
(B) Export kinetics of Arx1, Ebp1, and Map1 through the nuclear envelope of Xenopus oocytes. Arx1 shows facilitated diffusion through the NPCs,
whereas the human homolog Ebp1 and the methionine aminopeptidase Map1 are excluded from the microwells. The small and steep increase in the
beginning is ascribed to a low portion of free fluorophore.
We could solve the crystal structure of human Ebp1 at
a resolution of 1.6 Å (the structure coordinates of Ebp1
are available from PDB database under the accession
number 2Q8K; Kowalinski et al., 2007). Using the Ebp1
structure, we could obtain a structural model of the highly
related Arx1 by homology modeling. Accordingly, Arx1
shares the characteristic MetAP fold with central
b strands, which generate a deep cavity that resembles
the methionine substrate binding pocket in MetAPs (Figure 6). However, the critical residues of the active-site
pocket, which are involved in the proteolytic cleavage,
are changed in Arx1 (Figures 6D and 6E). Moreover, the
residues lining the hydrophobic methionine binding
pocket in MetAPs are different in Arx1 (Figure 6E). In addition, Arx1 exhibits several loop insertions, which are absent from MetAP (Figures 6A–6C). Taken together, this
analysis revealed that Arx1 has a MetAP fold with
a deep cavity that corresponds to the methionine substrate binding pocket in MetAPs, but the pocket residues
in Arx1 are different from MetAPs.
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The Central Cavity in Arx1 Is Involved in FG Repeat
Binding and Export of Pre-60S Subunits
These findings prompted us to test whether the pocket in
Arx1 could bind to FG repeats instead of methionine. Classical shuttling nuclear transport receptors (e.g., importins,
NTF2, and Tap-p15) have hydrophobic pockets/patches
on the protein surface that bind the FG peptide repeats
of FG nucleoporins (Bayliss et al., 2002). To determine
whether the central cavity of Arx1 is involved in FG repeat
interaction, we mutated nine residues that line this pocket
(either as single mutations or in combinations) to the respective amino acids in Ebp1 or to alanine (Figure 6E).
Moreover, an unrelated mutation was constructed in
which a long loop insertion of Arx1 (arx1Dloop 9) was deleted (Figures 6A and 6B). These altered Arx1 proteins
were expressed in yeast and tested for expression
(Figure 7A), genetic overlap with nmd3DNES1 (Figure 7B
and Figure S5A), binding to pre-60S particles and Alb1
(Figures S5B and S5C), subcellular location (Figure S5D),
and impairment of pre-60S subunit export (Figure 7C). A
few of these Arx1 mutants were also expressed in E. coli
to analyze the in vitro binding to FG repeat nucleoporins
(Figure 7D).
Single-pocket amino acid exchanges did not affect the
Arx1 function (Figure S5A; see also Figure 6E). However,
the arx1 dipocket2 mutant (W445F, R502Q) and its derived
tripocket mutant (I91A, W445F, R502Q) that were expressed normally in vivo (Figure 7A) caused a significant
growth inhibition when combined with the nmd3D NES1
mutation (Figure 7B). In contrast, another double-pocket
mutant, arx1 dipocket1 (I91A, R502Q), or the arx1Dloop
9 mutant did not exhibit a genetic link to nmd3DNES1 (Figure 7B). Consistent with these findings, both arx1 dipocket2 and arx1 tripocket, but not arx1 dipocket1 and
arx1Dloop 9, showed defects in 60S subunit export
when combined with ecm1D (Figure 7C), gle2D, or
nup116D (data not shown) and a reduced binding to
Nup100 FG repeats (Figure 7D). However, interaction
with pre-60S subunits and with Alb1 and the intracellular
location was not significantly altered in these arx1 mutants
(Figures S5B–S5D). Altogether, the mutational analyses
identified critical amino acids in the central cavity of Arx1
involved in the interaction with FG repeat nucleoporins
and nuclear export of pre-60S subunits.
DISCUSSION
Our studies have demonstrated that Arx1 is an additional
nuclear export receptor for the large ribosomal subunit.
Experimental evidence for this suggestion comes from
several directions. First, Arx1 is associated with the pre60S particle. It is not known to which factor(s) of the 60S
subunit Arx1 is bound, but candidates are ribosomal
RNA or ribosomal proteins (Hung and Johnson, 2006). Notably, MetAPs associate with mature 60S subunits in the
cytoplasm to remove N-terminal methionine from nascent
polypeptides (Vetro and Chang, 2002). Accordingly, Arx1,
which is homologous to MetAPs, could employ a related
mechanism to interact with the pre-60S subunit in the
nucleus.
Second, Arx1 can directly bind to the FG repeats of nucleoporins with dissociation constants in the low micromolar range. Classical shuttling transport receptors also
exhibit low-affinity binding constants for FG Nups (Bayliss
et al., 2002), which is the basis that these transporters can
pass through the ‘‘FG meshwork’’ of the active NPC transport channel (for review see Suntharalingam and Wente,
2003). Arx1 binds preferentially to the GLFG repeats of
Nup116 and Nup100. Preferred binding to subtypes of
FG Nups is also observed for classical nuclear transport
receptors (Pyhtila and Rexach, 2003). Thus, Arx1 may predominantly utilize the FG repeats of Nup116, Nup100, and
Nup42 for its translocation through NPCs.
Third, Arx1 exhibits facilitated translocation through the
NPCs. The velocity constant k = 0.004 s 1 for nuclear exit
of Arx1 in the Xenopus oocyte nuclear envelope translocation system corresponds to a transport rate of approximately eight translocation events per NPC per second.
Such kinetics would be compatible with estimated values
for yeast ribosome export, which is approximately one ribosome exported through one NPC every 3 s (Fatica and
Tollervey, 2002).
Figure 6. Structural Modeling of Arx1 Reveals a Methionine Aminopeptidase Fold
(A) Primary sequence alignment of yeast Arx1 with human Ebp1. The sequence alignment is supplemented by secondary structures, which are predicted for Arx1 (above sequences) and calculated (DSSP; Kabsch and Sander, 1983) from the Ebp1 structure (below sequences). Residues observed
in the crystal structure of Ebp1 or included in the Arx1 model are given in bold. Secondary structure elements are given in a color ramp from N- (blue) to
C terminus (red) and elements that are not modeled in gray. Conserved residues are colored in orange, and conservative exchanges in light orange.
Additional loop regions in Arx1 are marked in bold numbers (1–10) below the sequences. The deleted amino acid residues of arx1Dloop 9 (T465-K493)
are boxed, and the three residues mutated in arx1 tripocket are encircled (I91, W445, and R502).
(B) Homology model of Arx1 based on the Ebp1 structure. Color and numbers are corresponding to (A). Not modeled loop regions are denoted by
dashed lines and numbered as in (A).
(C) Methionine aminopeptidase structure. The crystal structure of human MetAP2 (PDB 1KQ9) is shown in the same color code as the Arx1 model in
(B) to highlight their similarity in structure. The structure contains two Zn2+ ions (purple) and a methionine (black) bound in the active site located within
the central cavity.
(D) Surface view of the models of Arx1 (left) and MetAP (right) as given in (B) and (C), respectively. Surface of residues in the area of the central cavity
are colored as in (E), left and right, respectively. Please note that the loop regions of Arx1 could not be modeled.
(E) Close-up view of the central cavity of Arx1 (left panel) with homologous residues important for metal and substrate binding in the respective
MetAP2 structures as shown in the right panel for human MetAP2. Most of the key residues are exchanged in Arx1, which explains the lack of methionine aminopeptidase activity. A special feature in Arx1 are four positively charged residues at the right side of the cavity (R243, R245, K322, and
R502) that are absent in MetAP2.
Molecular Cell 27, 767–779, September 7, 2007 ª2007 Elsevier Inc. 775
Molecular Cell
Arx1 Is a Nuclear Export Receptor for 60S Subunits
Figure 7. Predicted Pocket Residues in
Arx1 Are Crucial for FG Repeat Binding
and Nuclear Export of Pre-60S Subunits
(A) Expression of Arx1 mutant proteins in yeast.
Wild-type ARX1 and mutant alleles arx1D loop
9, arx1 dipocket1, arx1 dipocket 2, and arx1 tripocket were expressed in arx1D cells. Wholecell lysates were analyzed by SDS-PAGE and
western blotting using anti-TAP (Arx1) and
anti-Arc1 antibodies (loading control).
(B) Genetic link of arx1 pocket point mutations
with nmd3DNES1. The indicated arx1 mutant
alleles were expressed in the arx1D/NMD3
shuffle strain transformed with the respective
NMD3 alleles. Cells were spotted in serial 10fold dilutions on 5-FOA containing plates and
then incubated at 30 C for 4 days. The generated pocket point mutations are indicated on
the right.
(C) Analysis of nuclear export of 60S subunits
in arx1 pocket mutants. Strain ecm1D/arx1D
transformed with the indicated arx1 mutant alleles and expressing Rpl25-GFP was grown on
SDC at 30 C. Subcellular location of Rpl25GFP was analyzed by fluorescence microscopy.
(D) Binding of mutated Arx1 proteins to FG nucleoporins. Recombinant and purified wildtype and mutant Arx1 proteins were mixed
with E. coli lysate (load) and incubated with immobilized GST-Nup100 FG repeats. Bound
proteins were eluted from the beads with sample buffer. Load (lanes 1–5), bound (lanes 6–
10), and unbound proteins (lanes 11–15) were
analyzed by SDS-PAGE and western blotting
using the anti-pentaHis antibody to detect
Arx1. Lanes 1, 6, and 11, ARX1; lanes 2, 7,
and 12, arx1Dloop 9; lanes 3, 8, and 13, arx1 dipocket 1; lanes 4, 9, and 14, arx1 dipocket 2;
and lanes 5, 10, and 15, arx1 tripocket.
Fourth, in vivo Arx1 genetically overlaps with the known
components of the 60S subunit export machinery (e.g.,
Nmd3, Mex67-Mtr2). This observation raises the question
as to why the pre-60S subunit recruits more than one nuclear export receptor. It is known that the shuttling of
a transport receptor is slowed in proportion to increasing
cargo size (Ribbeck and Görlich, 2002). This implies that
large cargo may be hindered from passing through the
NPCs and require more than one receptor molecule for
rapid translocation. Pre-60S subunits are large assemblies in the range of 25–30 nm (Nissan et al., 2004).
Thus, pre-60S particles may recruit several export receptors for efficient passage through the pore channel
(Figure S6). After export to the cytoplasm, Arx1 was suggested to be released from the pre-60S particle by Rei1
(Hung and Johnson, 2006; Lebreton et al., 2006). Finally,
Arx1 may be reimported into the nucleus by its own shuttling activity or with the help of an import receptor (Lebreton et al., 2006).
Taking all the data together, we propose the following
model. Arx1 functions as an export receptor of the 60S
subunit in concert with the other exporters Xpo1 and
Mex67-Mtr2. Arx1 utilizes a cavity as part of the MetAP
fold to bind to the GLFG repeats of Nup116 and Nup100
instead of methionine. This interaction enables Arx1 to
pass through the NPCs with bound pre-60S cargo.
776 Molecular Cell 27, 767–779, September 7, 2007 ª2007 Elsevier Inc.
Molecular Cell
Arx1 Is a Nuclear Export Receptor for 60S Subunits
Notably, the human homolog of Arx1, a nucleolar and cytoplasmic protein with several names, including ErbB-3
receptor binding protein (Ebp1), proliferation-associated
2G4 protein (PA2G4), and IRES trans-acting factor
(ITAF45), exploits the MetAP fold and its central cavity in
a different way to perform diverse cellular functions such
as regulation of cell growth and differentiation, interaction
with transcription factors, ribosome biogenesis and rRNA
processing, and IRES-mediated translation (Pilipenko
et al., 2000; Xia et al., 2001; Squatrito et al., 2004, 2006).
Thus, the MetAP fold offers a versatile structure that enables members of the Arx1/Ebp1 protein family to perform
a wide range of diverse functions in eukaryotic cells.
EXPERIMENTAL PROCEDURES
Strains and Plasmids
Strains and plasmids used in this study are listed in Tables S1 and S2.
Mutants were generated by PCR-based approaches and verified by
DNA sequencing. Primer sequences are available upon request. The
synthetic lethal screen was performed according to Wimmer et al.
(1992).
Purification of Recombinant Proteins
Mex67-Mtr2 was purified according to Yao et al. (2007). GST-Nups
were purified essentially as described (Allen et al., 2001). His6-TEVlabeled Arx1 (wild-type or mutant), Ebp1, and Map1 were expressed
from plasmid pProEx1 in BL21 (DE3) Rosetta STAR E. coli. Cells
were lysed in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM b-mercaptoethanol + 0.1% Triton/0.5 mM PMSF, and a cocktail of protease
inhibitors. Lysates were incubated with Ni-NTA agarose (QIAGEN)
for 1 hr at 4 C. Beads were washed and eluted with imidazol.
For BIAcore or enzymatic activity measurements, Arx1, Ebp1, and
Map1 were further purified on Mono Q or Mono S columns. For labeling
with fluorescent dyes, the purified proteins were concentrated by ultrafiltration followed by buffer change (20 mM HEPES-KOH [pH 7.5],
110 mM KAc, 5 mM MgAc2, and 0.5 mM EGTA). Proteins were incubated with Alexa 488 fluorophor succinimidyl ester (Molecular Probes)
in an equimolar ratio for 2 hr on ice. Labeled proteins and unbound
fluorescent dyes were separated by Superdex200 gel filtration (Pharmacia) and Biogel P6 (Biorad) self-packed column.
MetAP Activity Enzymatic Assay
Methionine aminopeptidase activity measurements were performed
as described (Yang et al., 2001). The assay contained 50 mM TrisHCl (pH 7.5), 20 mM NaCl, 50 mM Amplex Red (Molecular Probes),
0.5U AAO (Worthington), 5U HRP (Fluka), and 0.4 mM L-Met-Ala-Ser
(Sigma). The reaction was started by addition of the recombinant protein. The emission at 590 nm was measured in Jasco FP-6500 spectrofluorometer (578 nm excitation).
In Vitro Binding of Arx1 to FG Nucleoporins
In vitro binding assay of Arx1 to FG repeat domains of nucleoporins
was performed as described (Sträßer et al., 2000). Recombinant purified protein was transferred to binding buffer (20 mM HEPES-KOH
[pH 6.8], 150 mM KOAc, 2 mM Mg[OAc]2, 0.1% Tween 20, 10% glycerol, and 2 mM DTT), centrifuged 30 min at 100,000 3 g, mixed with
E. coli lysate, and incubated for 2 hr at 4 C with 25 ml of GST-Nups
bound to glutathione-Sepharose 4B beads. The beads were washed
with binding buffer and bound proteins eluted with sample buffer.
BIAcore Binding Assay
Ligands (GST-Nup1, GST-Nup100) were immobilized via anti-GST antibody attached on CM5 research-grade sensor chips (BIAcore). Anal-
ysis of protein-protein interactions was carried out at a flow rate of
20 ml/min at 25 C with a BIAcore 2000 system as described (Katahira
et al., 2002). In parallel, purified His-tagged Arx1 at each concentration
(0.1–4.2 mM) was injected over a GST-immobilized flow cell to serve as
blank sensorgrams for background subtraction.
In Vitro Nuclear Import and Export Assays
In vitro import assay was performed essentially as described (Adam
et al., 1990; Imamoto et al., 1995). Nuclear export analysis by OSTR
was done according to Tschodrich-Rotter and Peters (1998), Kiskin
et al. (2003), and Peters (2003, 2006). In short, nuclei from Xenopus laevis oocytes were attached to a TC array (diameter 50 mm, depth 50 mm,
pitch 200 mm) and opened. Nuclear content was removed by rinsing
with mock3 buffer (90 mM KCl, 10 mM NaCl, 2 mM MgCl2, 0.1 mM
CaCl2, 1.0 mM HEDTA, and 10 mM HEPES [pH 7.3]). An area that contained nuclear envelope-covered and not covered TCs was imaged.
Fluorescence of Alexa 488-Arx1 (2.5–3.75 mM), Alexa 488-Ebp1 (2.7–
4 mM), or Alexa 488-Map1 (2.9 mM) and of control substrate dextran70
(2.5 mM) or Alexa 568-labeled MBP in 100 mM sucrose in mock3 was
imaged simultaneously every 6.3 s by laser scanning microscopy focused 20 mm below TC array surface. Transport rates were determined
with the ImageJ program (W. Rasband, http://rsb.info.nih.gov/ij/) with
multiple region-of-interest plug-in. Time-dependent intensity of each
TC was fitted by monoexponential curve using the LevenbergMarquardt algorithm (program Origin 7.0, OriginLab Corporation). Permeability coefficients and transport rates were calculated from TC
length and mean NPC density on nuclear membranes of 50 NPC/mm2.
Molecular Modeling of Arx1 Structure
The structure of Arx1 was obtained by homology modeling using the
solved structure of human Ebp1 (Kowalinski et al., 2007). Primary sequences were aligned by ClustalX, and regions not included in the
crystal structure were truncated. The alignment was locally corrected
where necessary with help of secondary structure predictions. Homology modeling was done with the program WHAT IF (Vriend, 1990),
which includes a refinement procedure for avoiding sterical clashes
and optimizing side-chain conformations.
Miscellaneous
Export of ribosomal subunits was analyzed according to Gadal et al.
(2001) and Milkereit et al. (2002). Poly(A)+ RNA location was analyzed
by in situ hybridization according to Santos-Rosa et al. (1998). TAP purification in standard buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl,
1.5 mM MgCl2, and 0.075% NP-40), GFP localization studies, western
analysis, and mass spectrometry were performed as described (Nissan et al., 2002). High-salt buffer for TAP-tag purification contained
110 mM HEPES-KOH (pH 7.8), 30 mM MgCl2, 400 mM (NH4)2SO4,
and 0.05% Triton X-100. Anti-TAP and anti-PentaHis antibodies
were purchased from BioCat and QIAGEN, respectively.
Supplemental Data
Supplemental Data include Supplemental References, six figures, and
three tables and can be found with this article online at http://www.
molecule.org/cgi/content/full/27/5/767/DC1/.
ACKNOWLEDGMENTS
The excellent technical assistance of Sabine Merker and Petra Ihrig
under the supervision of Dr. J. Lechner (Mass Spectrometry Unit,
BZH, Heidelberg) is acknowledged. The initial help of Dr. D. Mohr
with Arx1 import assay is gratefully acknowledged. We thank Drs. D.
Wolf, B. Stillman, and M. Groves for providing strains, Dr. M. Rexach
for GST-Nup expression plasmids, and Drs. C. Dargemont, A. Johnson, and J.R. Warner for antibodies. E.H. is a recipient of grants
from the Deutsche Forschungsgemeinschaft (Hu363/9-2 and Gottfried
Wilhelm Leibniz Program) and Fonds der Chemischen Industrie.
Molecular Cell 27, 767–779, September 7, 2007 ª2007 Elsevier Inc. 777
Molecular Cell
Arx1 Is a Nuclear Export Receptor for 60S Subunits
Received: January 20, 2006
Revised: March 30, 2007
Accepted: June 21, 2007
Published: September 6, 2007
Johnson, A.W., Lund, E., and Dahlberg, J. (2002). Nuclear export of
ribosomal subunits. Trends Biochem. Sci. 27, 580–585.
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Accession Numbers
The accession code of the Ebp1 structure is 2Q8K and is available
from the Protein Data Bank (PDB).
Molecular Cell 27, 767–779, September 7, 2007 ª2007 Elsevier Inc. 779