Molecular Biology of the Cell
Vol. 17, 5309–5323, December 2006
The Unfolded Protein Response Transducer Ire1p Contains
a Nuclear Localization Sequence Recognized by Multiple
Laurence Goffin,*†‡Sadanand Vodala,*†Christine Fraser,* Joanne Ryan,*
Mark Timms,* Sarina Meusburger,* Bruno Catimel,§Edouard C. Nice,§
Pamela A. Silver,?Chong-Yun Xiao,¶David A. Jans,¶#and Mary-Jane H. Gething*
*Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia;
§Ludwig Institute for Cancer Research, Parkville, Victoria 3052, Australia;?Department of Systems Biology,
Harvard Medical School, Boston, MA 02115;#Division of Biochemistry and Molecular Biology, John Curtin
School of Medical Research, Canberra, ACT 2601, Australia; and¶Department of Biochemistry and Molecular
Biology, Monash University, Clayton, Victoria 3800, Australia
Submitted April 11, 2006; Revised August 31, 2006; Accepted September 29, 2006
Monitoring Editor: Reid Gilmore
The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the
endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly
basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro.
This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process
requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin
binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS
(within sequences642KKKRKR647and/or653KKGR656) that is recognized by yeast importin ? (Kap60p) and a novel ?NLS
(646KRGSRGGKKGRK657) that is recognized by several yeast importin ? homologues. Kinetic binding data suggest that
binding to importin ? proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA
splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional
RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role
in UPR signaling.
Within the lumen of the endoplasmic reticulum (ER), a
variety of resident ER proteins assist newly translocated
nascent polypeptides to fold into their correct tertiary and
quaternary structures (Stevens and Argon, 1999). These res-
ident proteins include molecular chaperones that recognize
and stabilize partially folded intermediates during polypep-
tide folding and assembly, as well as enzymes that catalyze
rate-determining steps in folding, such as protein disulfide
isomerase and peptidyl prolyl isomerases. Under normal
growth conditions these chaperones and folding catalysts
are synthesized constitutively and abundantly. However,
their rates of synthesis can be increased significantly by the
accumulation of mutant proteins in the ER or by a variety of
stress conditions whose common denominator is thought to
be the accumulation in the ER of unfolded polypeptides
(Kozutsumi et al., 1988; Mori et al., 1992). This “unfolded
protein response” (UPR) operates in yeast and higher eu-
karyotes to regulate the levels of ER chaperones and protein
folding catalysts (for review, see Ma and Hendershot, 2001;
Patil and Walter, 2001; Kaufman, 2002; Ron, 2002). Microar-
ray analysis of yeast cells demonstrated that the UPR also
activates genes encoding a variety of other proteins involved
in diverse processes such as translocation, glycosylation and
degradation of secretory proteins, lipid/inositol metabo-
lism, cell wall biogenesis, vesicle trafficking/transport, and
vacuolar protein sorting (Travers et al., 2000). Conserved
elements (UPREs) are present in the promoter regions of
many UPR-regulated yeast genes (Mori et al., 1992, 1998;
Patil et al., 2004). Thus, an intracellular sensing system mon-
itors events in the lumen of the yeast ER and transduces
signals across the ER membrane and into the nucleus to
activate the transcription of UPRE-controlled genes.
In Saccharomyces cerevisiae, the ER-to-nucleus (ERN) signal
transduction pathway contains two unique components, the
Ire1p/Ern1p transmembrane protein and the bZIP Hac1p
transcription factor that binds UPREs. These components
are not essential for vegetative growth, but they are abso-
lutely necessary for survival under conditions that cause
UPR stress (Cox et al., 1993; Mori et al., 1993). Ire1p contains
in one molecule three of the essential components of the
UPR pathway: the lumenal sensor, the mechanism for trans-
ducing the signal across the ER membrane, and the mecha-
This article was published online ahead of print in MBC in Press
on October 11, 2006.
†These authors contributed equally to this work.
‡Present address: Department of Tumor Immunology, Ludwig In-
stitute for Cancer Research, Lausanne 1066, Switzerland.
Address correspondence to: Mary-Jane H. Gething (m.gething@
© 2006 by The American Society for Cell Biology5309
nism of transcriptional activation of UPRE-controlled genes.
The glycosylated N-terminal portion of Ire1p is located in
the ER lumen, and, apparently through binding to uncom-
plexed BiP (Kohno et al., 1993; Okamura et al., 2000), senses
the load of misfolded proteins within the ER. The C-terminal
half of Ire1p carries an essential protein kinase domain (Mori
et al., 1993), which is activated by receptor dimerization
(Shamu and Walter, 1996; Welihinda and Kaufman, 1996), as
well as a C-terminal domain that functions as an RNA
endonuclease after activation by UPR stress (Sidrauski and
Walter, 1997). This endoribonuclease, together with Rlg1p
(previously identified as a tRNA ligase) (Sidrauski et al.,
1996), is required for the splicing of the mRNA that encodes
Hac1p (Cox and Walter, 1996; Mori et al., 1996). Additional
participants in the UPR response in yeast include the Ada2p
and Ada5p subunits of the Gcn5 transcriptional coactivator
complex, which interact with sequences within the C-termi-
nal half of Ire1p (Welihinda et al., 2000), and the Gcn4p
transcriptional activator, which cooperates with Hac1p to
transactivate UPRE-containing genes (Patil et al., 2004).
The ERN signaling pathway outlined above presents a
topological problem because it suggests that the C-terminal
domain of Ire1p functions in the nucleus, where the Rlg1p
ligase (Clark and Abelson, 1987) and the components of the
Gcn5 complex are located. Thus, if Ire1p functions as an
intact transmembrane protein (Shamu and Walter, 1996; see
data below), it must be localized to the inner nuclear mem-
brane, as has been demonstrated for the murine Ire1? ho-
mologue of yeast Ire1p (Lee et al., 2002). However, Ire1p,
which contains an N-terminal hydrophobic signal sequence
for targeting to the ER (Mori et al., 1993), must be synthe-
sized on membrane-bound ribosomes that are present on the
rough ER, which includes the outer nuclear membrane, but
that are not present on the inner nuclear membrane. The
inner and outer nuclear membranes are separated by nu-
clear pores that control the flow of macromolecules into and
out of the nucleus (Rout et al., 2000). The signal-dependent
trafficking of proteins and RNA species through these pores
mediated by importins ? and ? is now well documented
(Go ¨rlich and Kutay, 1999; Strom and Weis, 2001; Poon and
Jans, 2005), but very little is known about translocation of
membrane proteins between outer and inner nuclear mem-
branes. Here, we demonstrate that a highly basic sequence
within the linker region of Ire1p can mediate nuclear local-
ization of green fluorescent protein and is recognized by
both importin ? and multiple importin ? proteins. Muta-
tions within this sequence that prevent the accumulation of
Ire1p in the nucleus result in impaired processing of HAC1
mRNA and inhibition of the UPR. This work provides the
first evidence that Ire1p interacts with the nuclear import
machinery and suggests that nuclear localization of Ire1p is
essential for UPR signaling.
MATERIALS AND METHODS
Strains and Plasmids
Descriptions and sources of the strains and plasmids used in this study are
listed in Table 1. Yeast cells were grown in YP medium containing glucose
(YPD) or synthetic complete media lacking appropriate amino acids (Kaiser et
al., 1994). Strains containing a temperature-sensitive mutation were grown at
Table 1. List of yeast strains and plasmids
MAT? leu2-3,112 ura3-52 his3?200 trp1?901 lys2-801
MAT? leu2-3,112 ura3-52 his3?200 trp1?901 lys2-801 sec53-6
KMY1005 ern1?::TRP1 UPRE-lacZ::URA3
KMY2005 ern1?::TRP1 UPRE-lacZ::URA3
MATa ura3-52 leu2?1 trp1?63
MAT? ura3-52 leu2?1 trp1?63 his3?200 ade2?::hisG srp1-31
MAT? ura3-52 leu2?1 trp1?63 prp20-1
MAT? ura3-52 leu2?1 ade2?::hisG rna1-1
MAT? ura3-52 leu2?1 ade2?::hisG ade3 rna1-1 (2?-ADE3-RNA1)
MATa ura3-52 leu2?1 trp1?63 his3?200 gsp1::HIS3(CEN-LEU2-gsp1-1)
MAT? ura3-52 leu2?1 his3?200 kap123?::HIS3
MATa ura3-52 trp1?63 his3?200 pse1-1 kap123?::HIS3
MATa ura3-52 leu2?1 trp1?63 rsl1-4
MAT? ura3-52 leu2?1 his3?200 ade2?::hisG ade8?100::KANRnmd5?::HIS3
MATa ura3-52 leu2?1 his3?200 trp1?63 lys2 sxm1?::HIS3
MATa ura3-52 leu2?1 trp1?63 pse1-1
PSY580 with integrated KAP108-GFP replacing KAP108 (SXM1)
PSY580 with integrated KAP114-GFP replacing KAP114
PSY580 with integrated KAP95-GFP replacing KAP95 (RSL1)
PSY580 with integrated KAP104-GFP replacing KAP104
PSY580 with integrated KAP111-GFP replacing KAP111 (MTR10)
PSY580 with integrated KAP123-GFP replacing KAP123 (YRB4)
PSY580 with integrated KAP121-GFP replacing KAP121 (PSE1)
MATa ura3 leu2 his3 lys2 ire1?::kanMX
P. A. Silver
P. A. Silver
P. A. Silver
P. A. Silver
2?-based yeast vector, LEU2, ERN1 (IRE1) under ERN1 promoter
CEN6-ARS1-based yeast vector, LEU2, ERN1 under KAR2 promoter
CEN6-ARS1-based yeast vector, URA3, GFP under MET promoter
The references cited are as follows: 1. Mori et al. (1996); 2. Kawahara et al. (1997); 3. Winston et al. (1995); 4. Loeb et al. (1995); 5. Koepp et al.
(1996); 6. Wong et al. (1997); 7. Seedorf and Silver (1997); 8. Ferrigno et al. (1998); 9. Aitchison et al. (1996); 10. Seedorf et al. (1999); 11.
Morehouse et al. (1999); 12. Mori et al. (1993); 13. Niedenthal et al. (1996). Note that ern1? ? ire1?.
L. Goffin et al.
Molecular Biology of the Cell 5310
the permissive temperature (23°C) before incubation under nonpermissive
conditions (37°C). All plasmid manipulations were carried out using standard
protocols (Sambrook et al., 1989). The sequences of oligonucleotides used in
this study are available upon request. DNA sequence analysis was used to
confirm the accuracy of introduced mutations.
Mutations in ERN1/IRE1 encoding alterations in the classical nuclear localiza-
tion signal (cNLS) were generated by oligonucleotide-mediated site-directed
mutagenesis (Kunkel, 1985). Restriction fragments encompassing the mutated
sequences were inserted into both the pERN1EM and pERN1BC plasmids,
replacing the corresponding wild-type sequence. Plasmids capable of expressing
GFP–Ire1p fusion proteins under the control of the MET25 promoter were
constructed as follows: Nucleotides encoding the C-terminal region of Ire1p
(residues 556–1115) were amplified by polymerase chain reaction (PCR) by using
oligonucleotides VS1 and VS2 as primers and pERN1BC3 as the template. The
primers also added terminal SpeI and XhoI restriction sites that were used to
clone the amplified fragments between the SpeI and XhoI sites of pGFP-N-FUS
to generate pGFIC. Plasmid pGFIC(K646A,R647A) was created from pGFICusing
the complementary mutagenic oligonucleotides VS3 and VS4 and the
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Plasmid
pISGFFVITMCwas created from pGFICby inserting an XbaI–SpeI fragment
from pC4-FV1E (Ariad Pharmaceuticals, Cambridge, MA) that encodes a
variant FKBP (Spencer et al., 1993; Clackson et al., 1998) between the XbaI and
SpeI sites of pGFIC, then inserting a double-stranded oligonucleotide (VS5/
VS6) encoding the Ire1p transmembrane domain (residues 525–555) into the
SpeI site of the resulting construct (pGFFVIC) to generate pGFFVITMC, and
finally by inserting another double-stranded oligonucleotide (VS7/VS8) en-
coding the first 26 residues of Ire1p, which includes the ER targeting se-
quence, into the EcoR1 site of pGFFVITMCto generate pISGFFVITMC. DNA
sequence analysis was used to confirm the correct insertion and orientation of
the introduced nucleotide sequences at each step. The same procedures were
used to generate pISGFFKITMC(K646A, R647A) from pGFIC(K646A, R647A). Nu-
cleotides encoding wild-type or mutant versions of the linker region of Ire1p
(residues 556–672) were amplified by PCR by using oligonucleotides VS1 and
VS9 as primers and pERN1BC3 or pERN1BC(K644T,R645T) as templates. The
primers also added terminal SpeI and XhoI restriction sites that were used to
clone the amplified fragments into pGFP-N-FUS to give rise to pGFILand
pGFIL(K644T,R645T), respectively. To create the pGFIBseries of plasmids, pairs
of complementary oligonucleotides (VS10–VS19), which form duplexes en-
coding either wild-type or mutant versions of the 19 residue NLS (residues
642–660) flanked by SpeI and XhoI restriction sites, were phosphorylated
using T4 polynucleotidyl kinase. Each complementary pair was then allowed
to anneal in an Eppendorf thermocycler by using a program with a temper-
ature drop of 5°C every 5 min starting from 95°C and ending at 23°C. The
annealed duplex DNAs were purified on 15% nondenaturing polyacrylamide
gels and cloned between the SpeI and XhoI sites of the pGFP-N-FUS vector.
To generate pLG316, the KAP123 gene was amplified by PCR. The introduc-
tion of two flanking SmaI restriction sites within the oligonucleotide primers
(LG1 and LG2) enabled in-frame subcloning of the KAP123 open reading
frame (ORF) into the SmaI restriction site at the 3? end of the glutathione
S-transferase (GST) gene in pGEX-2TK.
ENLS peptides (see sequences in Figures 4A and 9) were synthesized by
continuous flow solid-phase synthesis by using fluorenylmethoxycarbonyl- or
tert-butyloxycarbonyl-protected amino acids, purified using a model 1100
liquid chromatograph (Hewlett Packard, Palo Alto, CA) with a Hypersyl ODS
micropreparative reverse phase-high performance liquid chromatography
column (Hewlett Packard) and analyzed by matrix-assisted laser desorption
ionization/time of flight mass spectrometry (Kratos Analytical, Manchester,
Lancashire, United Kingdom) and amino acid analysis. The importin ?1-
recognized peptide representing parathryoid hormone related protein
(PTHrP) residues 67–94 (YLTQETNKVETYKEQPLKTPGKKKKGKP) has
been described previously (Lam et al., 1999).
Expression of GST Fusion Proteins
Expression and purification of murine (m-PTAC58/Rch1 and PTAC97) or
yeast (Kap60p or Kap95p) importin ? and ? subunits fused to the GST protein
were performed as described previously (Lam et al., 1999). Human Ran was
also expressed as a GST fusion protein (Hu and Jans, 1999) and then GST-free
Ran was prepared by thrombin cleavage and loaded with nucleotides as
described by Chi et al. (1996). All purified proteins were dialyzed against
storage buffer (20 mM HEPES, pH 7.3, 100 mM KOAc, and 2 mM dithiothre-
itol [DTT]) and kept frozen at ?80°C until use.
Enzyme-linked Immunosorbent Assay (ELISA)-based
Importin Binding Assay
Binding of GST-importin fusion proteins to Ire1p peptides was quantitated
using an ELISA-based assay (Hu and Jans, 1999). Where inhibition studies
were performed, importin and Ran proteins were added simultaneously to
the immobilized peptide. The amount of bound importin was determined
using an antibody directed against the GST moiety of the importin fusion
protein (goat anti-GST antibody; GE Healthcare, Little Chalfont, Buckingham-
shire, United Kingdom).
Biosensor-based Importin Binding Assay
Biosensor analyses were performed using an optical biosensor (BIAcore 2000;
BIAcore, Uppsala, Sweden) (Nice and Catimel, 1999). To obtain a defined
orientation of peptide on the sensor surface and achieve optimum binding
presentation for the importin proteins, the ENLS-1 peptide was thiol conju-
gated via the N-terminal cysteine on the sensor surface (Catimel et al., 1997).
Binding data were generated by passing varying concentrations of GST–
importin fusion proteins over the immobilized peptide.
Binding of Importin ?-Green Fluorescent Protein (GFP)
Fusion Proteins to Resin-linked NLS Peptides
ENLS-4 peptides (see Figure 9 legend for sequence) were linked via their
N-terminal cysteine residues to thiopropyl-Sepharose 6B as described by the
manufacturer (Pharmacia, Uppsala, Sweden). Aliquots of peptide-linked
resin (25 ?l of packed resin suspended in 50 ?l of 0.1 M Tris-HCl, pH 7.0, 1
M NaCl, and 1 mM EDTA [EQ buffer]) were incubated for 1 h on ice with
50-?l aliquots of yeast cell extracts containing importin ?-GFP fusion proteins,
prepared as described by Seedorf et al. (1999). The unbound fraction was
separated from resin by centrifugation in a Microfuge, and the resin was then
washed three times with EQ buffer containing 0.5% Triton X-100 before bound
importin-GFP proteins were eluted from the resin with 0.1 M Tris-HCl, pH
8.3, 1 M NaCl, 1 mM EDTA, and 20 mM DTT. In some experiments, lysates
were incubated in the presence of 1 mM guanosine 5?-O-(3-thio)triphosphate
(GTP?S) or GDP (Sigma-Aldrich, St. Louis, MO) before the peptide resin was
Cell Extracts and Immunoblotting
Protein extracts from yeast cells were made using EZ buffer [60 mM Tris-HCl,
pH 6.8, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 5% (vol/vol) ?-mer-
captoethanol] and quantitated using the Bradford protein assay kit (Bio-Rad,
Hercules, CA). Volumes of extract containing equal amounts of total protein
(?150–200 ?g) were boiled in 5X SDS sample buffer for 10 min and then
loaded onto 8 or 12% acrylamide gels for PAGE. For immunoblot analysis,
proteins were transferred onto nitrocellulose (0.45-?m Protran; Whatman
Schleicher and Schuell, Dassel, Germany) via wet transfer in the Mini Trans-
Blot Cell (Bio-Rad) and blocked with 5% skim milk in Tris-buffered saline
containing 0.1% Tween 20. Detection of Ire1p was performed using a poly-
clonal antibody directed against residues 32–259 of Ire1p/Ern1p (Mori et al.,
1993), and GFP fusion proteins were detected using polyclonal anti-GFP
antibodies (Seedorf et al., 1999). Subsequently, the membranes were probed
with monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(a gift from Trevor Lithgow, University of Melbourne, Melbourne, Australia)
as a control for protein loading. The proteins were detected using ECL
reagents (Pierce Chemical, Rockford, IL, or Roche Diagnostics, Mannheim,
Germany) according to the manufacturer’s instructions.
?-Galactosidase activity was assayed by the crude extract method of Kaiser et
al. (1994). Protein concentrations were determined using the DC Protein
Assay kit (Bio-Rad).
HAC1 mRNA Splicing
Total cellular RNA from intact yeast cells was isolated as described by
Ausubel et al. (1995). Total RNA (5.4 ?g) was denatured using glyoxal and
separated by electrophoresis on a 1.4% agarose gel. Separated RNA species
were transferred onto nitrocellulose membrane (Nylon N?; GE Healthcare)
and probed with random primer32P-labeled HAC1 PCR products. Autora-
diography was performed either at room temperature or ?70°C. Experimen-
tal details are reported in the figure legends.
Yeast Fluorescence Microscopy
Yeast cells expressing GFP fusion proteins were prepared for visualization as
follows: log-phase cultures (OD600? 0.6–0.8) were grown at 30°C in synthetic
medium in the absence of methionine and uracil to induce expression from
the MET25 promoter of pGFP-N-FUS vector. When the expressed fusion
proteins contained the FKBP-based dimerization sequence (FV) dimerization
domain (Spencer et al., 1993; Clackson et al., 1998), the cell-permeant dimerizer
AP20187 (Ariad Pharmaceuticals) was added to the medium at a final con-
centration of 2 ?M, and incubation was continued for 2 h. Cells were fixed by
adding 1/10 volume of a 37% stock solution of formaldehyde directly to the
medium and incubating for at least 30 min. Harvested cells were washed
twice with 0.1 M potassium phosphate, pH 7.5, followed by two washes with
1X phosphate-buffered saline (PBS), and then resuspended in PBS (200 ?l per
10 ml of culture). Twenty-microliter aliquots of cell suspensions were
smeared evenly on cover slips coated with poly-l-lysine and allowed to dry.
The coverslips were then inverted onto a 10-?l drop of Mowiol containing 5
ng/?l 4,6-diamidino-2-phenylindole (DAPI) on a glass slide. Fluorescence
Nuclear Localization Sequence in Ire1p
Vol. 17, December 20065311
microscopy was carried out using either an Axioplan 2 imaging system (Carl
Zeiss, Jena, Germany) and picture analysis using the Axioplan 3.0 software
(Carl Zeiss), or an Olympus IX70 inverted microscope with picture analysis
using Openlab software (Improvision, Lexington, MA). When desirable, flu-
orescence images were deconvoluted using Volocity software 3.7.0. Data sets
for two channels were captured using GFP and DAPI filters. Exposure for the
green channel was for 300 ms, whereas that for the blue DAPI channel was for
150 ms. Iterative deconvolution microscopy was performed on Z-stacks of 61
images with a step size of 0.2 ?m. Significance was determined using 99.5%
confidence limit or 25 iterations.
The Linker Region of Ire1p Contains a Nuclear
The kinase and endonuclease domains of Ire1p do not contain
any clusters of basic residues that might act as NLSs. However,
as noted previously (Mori et al., 1993) and shown in Figure 1A,
the 117-residue linker region (residues 556–672) contains a
highly basic sequence (residues 642–659, underlined) that in-
cludes a stretch of six residues (642KKKRKR647) that closely
resemble the paradigmatic cNLS of SV40 large T antigen
(127KKKRKV132; Dingwall and Laskey, 1991), as well as six
additional lysine and arginine residues just downstream from
To determine whether the Ire1p linker sequence contains a
functional NLS that could direct the nuclear localization of the
GFP reporter, we constructed various plasmids expressing
GFP–Ire1p fusion proteins, under the control of the MET25
promoter. The GFP moiety was fused either to the entire C-
terminal domain (GFIC; Figure 1A, ii) or to the linker region
(GFIL; Figure 1A, iii). The parental plasmid that expresses un-
fused GFP was used as the negative control. The pGFP, pGFIC,
or pGFILplasmids were introduced into RG11907 yeast cells,
and the expression levels of GFP and GFP fusion proteins were
analyzed by immunoblotting with an anti-GFP antibody (Fig-
ure 1B), whereas their intracellular localizations were analyzed
by fluorescence microscopy (Figure 1C). Cells expressing un-
fused GFP displayed an even distribution of fluorescence (Fig-
ure 1C, first column, top), with the exception of a dark patch
that does not colocalize with the nucleus (shown by DAPI
staining, middle), but probably corresponds to the vacuole.
The attachment of the entire C-terminal domain or just the
linker sequence caused accumulation of GFP in the nucleus
(Figure 1C, second and third columns). We concluded that the
C-terminal domain of Ire1p contains a functional NLS that is
likely to involve basic amino acids located between residues
642 and 659.
The potential cNLS in the basic sequence contains three
overlapping versions of the cNLS consensus motif (K.R/
K.x.R/K, Fontes et al., 2000). To determine whether one or
more of these motifs is essential for nuclear targeting, a
mutant version of pGFP-Ire1Lwas constructed in which
threonine residues were substituted for K644and R645in the
encoded fusion protein. These two amino acid substitutions
interrupt all three versions of the cNLS motif and corre-
spond to mutations that individually abolish or diminish the
activity of the T-ag cNLS (Kalderon et al., 1984). Cells ex-
pressing the GFP-Ire1L(K644T,R645T) fusion protein (Figure
1B, see immunoblot in right-hand panel) showed greatly
reduced nuclear fluorescence, which occurred at a level only
slightly higher than that in the surrounding cytoplasm (Fig-
ure 1C, right-hand column). We concluded that residues
K644and R645are important but not absolutely essential for
nuclear accumulation of the fusion protein.
Mutation of the Potential cNLS in Ire1p Inhibits the UPR
region are required for UPR signaling, site-directed mutagen-
localization of GFP. (A) Ire1p is a 1115-amino acid transmembrane
protein that contains a classic N-terminal hydrophobic signal se-
quence for ER localization. The various domains of the protein are
indicated in i. GFP-fusion proteins containing the C-terminal do-
main (GFIC) or the linker sequence (GFIL) of Ire1p are shown in ii
and iii, respectively. Shown in iv is the sequence of the linker
domain, which contains a stretch of basic residues (in bold) that re-
sembles the cNLS (PKKKRKV) of SV40 large T-antigen. This sequence
is part of an 18-residue sequence (underlined) that is very rich in basic
residues. (B) Equal amounts of protein extracts prepared from yeast
cells expressing GFP or GFP-Ire1 fusion proteins were resolved by
SDS-PAGE and analyzed by immunoblotting using anti-GFP and anti-
GAPDH antibodies. (C) Single colonies of RG11907 yeast cells freshly
transformed with plasmids encoding GFP or GFP–Ire1 fusion proteins
were grown to an OD600of 0.6 in SC-URA-MET medium before
examination of the cells by fluorescence microscopy.
Sequences in the Ire1p linker region promote nuclear
L. Goffin et al.
Molecular Biology of the Cell5312
esis of the cDNA encoding full-length Ire1p was used to sub-
stitute threonine residues for K644, or for both K644and R645, or
to delete all six basic residues. The single K644T substitution
leaves intact only the first of the three versions of the cNLS
motif, whereas the deletion mutant, like the double substitu-
tion mutant, lacks all three versions. The wild-type and mu-
tated Ire1 proteins were expressed in ire1? (ern1?) yeast cells
by using single copy (CEN) or multicopy (2?) vectors, and ER
stress was imposed by treatment of cultures with tunicamycin,
a drug that blocks protein glycosylation and causes the accu-
mulation of unfolded proteins in the ER. Immunoblotting of
protein extracts of unstressed and stressed cells demonstrated
that the mutant proteins were expressed at levels equivalent to
that of wild-type Ire1p (Figure 2A). No cleavage of the Ire1
polypeptides could be discerned after stress treatment, con-
firming previous data (Shamu and Walter, 1996) that UPR
signaling does not involve proteolytic release of the kinase and
The UPR response in cells expressing either the wild-type
or mutated Ire1 proteins was measured by analysis of ?-ga-
lactosidase activity in cells containing a UPRE-controlled
lacZ reporter gene (Mori et al., 1993). As shown in Figure 2B,
the single K644T substitution had little or no effect on signal-
ing by Ire1p, but substitution of both K644and R645by
threonine caused a significant (90%) decrease in the UPR
response. This phenotype is consistent with the significant
reduction observed in the nuclear targeting activity of the
GFP-Ire1Lprotein containing the same amino acid substitu-
tions (see above; Figure 1C). ERN signaling was essentially
absent in cells expressing the ?NLS mutant, suggesting ei-
ther that one or more of the other basic residues in the
putative cNLS are required in addition to residues K644and
R645for maximal UPR signaling or that deletion of all six
residues abolishes signaling as the result of misfolding of the
mutant protein. However, the data are not compatible with
some or all of residues 642–647 constituting a cNLS that is
essential for the function of Ire1p because the double sub-
stitution mutant still retains measurable signaling activity
(?10%), despite lacking a cNLS motif. We therefore consid-
ered the possibility that the functional NLS involves addi-
tional residues within the extended basic sequence (residues
642–659) of the Ire1 linker (Figure 1A).
The Ire1p NLS Is an Extended Basic Sequence
Plasmids were constructed that encode GFP fused to Ire1p
residues 642–659 (GFP-Ire1B), or mutant versions of this
basic sequence in which lysine and arginine residues that lie
downstream of R645were substituted, either singly or in
pairs, by alanine (Figure 3A). K642 and K643 were not
targeted because parallel in vitro peptide binding studies
described below had demonstrated that these residues were
sequence inhibit nuclear localization. (A) Residues mutated within
the Ire1p linker sequence in GFP-Ire1Bfusion proteins are under-
lined. (B) Equal amounts of protein extracts prepared from yeast
cells expressing GFP or GFP-Ire1Bfusion proteins were resolved by
SDS-PAGE and analyzed by immunoblotting using anti-GFP and
anti-GAPDH antibodies. (C) Single colonies of RG11907 yeast cells
freshly transformed with plasmids encoding GFP or GFP-Ire1Bfu-
sion proteins were grown to an OD600of 0.6 in SC-URA-MET
medium before examination of the cells by fluorescence microscopy.
Point mutations of basic residues in the Ire1p linker
mutant cNLS. (A) Wild-type and mutant Ire1p/Ern1p proteins were
expressed in KMY2115 ern1? yeast cells from multicopy (2?)
pERN1-EM vectors (Mori et al., 1993). Mid-log cultures were incu-
bated for 3 h at 23°C in the presence or absence of tunicamycin (Tu;
5 ?g/ml final). Proteins were then extracted, and equal amounts of
total proteins (measured by Bradford DC assay; Bio-Rad) were
resolved by SDS-PAGE and analyzed by immunoblotting by using
the anti-N1 Ire1p-specific primary antibody. (B) KMY1115 ern1?
yeast cells containing wild-type or mutant Ire1 proteins expressed
from single copy (CEN) pERN1-BC vectors (Mori et al., 1993) were
grown to mid-logarithmic phase and then incubated at 30°C for 3 h
in the presence (closed bars) or absence (open bars) of Tu (5 ?g/ml
final). Protein extracts were prepared and assayed for ?-galactosi-
dase activity by using a UPRE-controlled lacZ reporter gene (Mori et
al., 1992). The results are presented as mean ? SD, based on dupli-
cate determinations with three independent transformants and are
normalized to 100% of the activity obtained with wild-type Ire1p.
UPR signaling in cells expressing Ire1 proteins with
Nuclear Localization Sequence in Ire1p
Vol. 17, December 2006 5313
not required for binding to importin proteins. The pGFP-
Ire1Bplasmids were introduced into RG11907 yeast cells,
and the expression levels of the fusion proteins were ana-
lyzed by immunoblotting with an anti-GFP antibody (Figure
3B). The intracellular localization of GFP was again analyzed
by confocal microscopy (Figure 3C).
Cells expressing GFP fused to the wild-type Ire1p basic
sequence displayed bright nuclear fluorescence (Figure 3C,
second column), confirming that this sequence functions as an
efficient NLS. All of the substitution mutations caused reduced
accumulation of the fusion proteins in the nucleus, although
the extent of the reduction varied. The K646A,R647A and
R656A,K657A double mutations almost completely prevented
nuclear accumulation (Figure 3C, third and sixth columns),
whereas the K653A,K654A double mutant, which consistently
accumulated to lower levels than the other fusion proteins
(Figure 3B), displayed both cytoplasmic and nuclear localiza-
tion (Figure 3C, fifth column). The R650A mutant, although still
predominantly localized in the nucleus, displayed significantly
more cytoplasmic fluorescence than the construct containing
the wild-type basic sequence (Figure 3C, fourth column). To-
gether, these data define a minimal NLS consisting of 13 resi-
Components of the Ran Cycle Are Required for HAC1
mRNA Splicing and for Import of Ire1p NLS-containing
To determine whether components of the nuclear localiza-
tion apparatus are essential for the early events of UPR
signaling, we tested whether mutations in proteins involved
in the Ran cycle required for the orientation of importin-
mediated nuclear transport (reviewed by Go ¨rlich and Kutay,
1999) would affect ER stress-induced HAC1 mRNA splicing.
We assayed HAC1 splicing rather than the induction of
UPRE-controlled genes because the overall UPR signaling
pathway requires two additional nuclear transport steps
(export of HAC1 mRNA and import of the translated Hac1p
transcription factor) that also might be affected in these
mutants. Significant levels of stress-induced HAC1 mRNA
splicing were observed at 23 and 37°C in wild-type yeast
cells (strain PSY580; Figure 4A, first panel). Similar levels of
splicing occurred at the permissive temperature of 23°C in
cells carrying temperature-sensitive (ts) mutations in the
genes encoding RanGTPase itself (gsp1? expressing gsp-1
strain PSY961), RanGAP (rna1-1 strain PSY868), and RanGEF
(prp20-1 strain PSY713). However, after incubation at the
nonpermissive temperature of 37°C, HAC1 mRNA splicing
was significantly reduced in gsp-1 cells and completely in-
hibited in rna1-1 and prp20-1 cells.
To test whether the nuclear targeting activity of the Ire1p
NLS is dependent on a functional Ran cycle, the pGFP,
pGFP-Ire1C, and pGFP-Ire1C(K646A,R647A) plasmids were
introduced into PSY580 and PSY868 (rna1-1) cells. The ex-
pression levels (Figure 4B) and intracellular localizations
(Figure 4C) of GFP and the GFP fusion proteins were ana-
lyzed after growth at 23 or 37°C. The growth temperature
had little or no effect on the expression levels of the GFP
proteins (Figure 4B), and it did not alter the cellular local-
ization of these proteins in the parental PSY580 cells. How-
ever, in rna1-1 cells the GFICfusion protein was predomi-
nantly localized to the nucleus after incubation at 23°C, but
it was redistributed to the cytoplasm after incubation at
37°C. By contrast, the largely cytoplasmic localizations of
unfused GFP and the GFIC(K646A,R647A) fusion protein were
required for HAC1 mRNA splicing and for
import of Ire1p NLS-containing proteins. (A)
ER stress-induced HAC1 mRNA splicing was
assayed in wild-type yeast (strain PSY580) and
strains carrying ts mutations in GSP1 (RanGT-
Pase), RNA1 (RanGAP), or PRP20 (RanGEF).
Cultures were grown at 23°C to an OD600of 0.6
and then split into two aliquots, one of which
was shifted to 37°C. After incubation for 4 h,
tunicamycin was added to both cultures to a
final concentration of 5 ?g/ml, and a sample
was taken immediately (?Tu) and snap-frozen
before incubation was continued at 23 or 37°C.
One hour later, a second sample (?Tu) was
taken from each culture. Total RNA was ex-
tracted from each sample as described by
Ausubel et al. (1995). After electrophoresis of
glyoxal-denatured RNA through a 1.4% aga-
rose gel containing 10 mM iodoacetic acid, the
RNA was blotted to NYTRAN Plus, probed
with randomly primed32P-labeled HAC1 PCR
products, and autoradiographed. The relative
migration of the various HAC1 mRNA species
is indicated on the right of the panel (u, un-
spliced; s, spliced). (B) PSY580 or PSY868
(rna1-1) yeast cells expressing GFP or GFP-
Ire1Cfusion proteins were grown at 23°C to an
OD600of 0.6 and then split into two aliquots,
one of which was shifted to 37°C. After incu-
bation for 4 h, protein extracts were prepared,
resolved by SDS-PAGE, and analyzed by im-
Components of the Ran cycle are
munoblotting by using anti-GFP and anti-GAPDH antibodies. (C) Single colonies of PSY580 or PSY868 (rna1-1) yeast cells expressing GFP
or GFP-Ire1Cfusion proteins were grown at 23°C to an OD600of 0.6 in SC-URA-MET medium and then split into two aliquots. One aliquot
remained at 23°C, whereas the second aliquot was shifted to 37°C. After incubation for 4 h, the cells were examined by fluorescence
L. Goffin et al.
Molecular Biology of the Cell5314
not affected by the change in temperature. Essentially iden-
tical results were obtained when these experiments were
repeated in PSY713 (prp20-1) cells (data not shown). To-
gether, these results indicate that a functional Ran cycle is
required for the nuclear targeting activity of the Ire1p NLS
and for HAC1 mRNA splicing by Ire1p.
Membrane-anchored Ire1p Sequences Are Targeted to the
Ire1p is normally present in yeast cells at such low levels
(Mori et al., 1993) that the wild-type protein cannot be visu-
alized by immunocytochemistry. A perinuclear and periph-
eral ER localization pattern has been reported for an Ire1p–
GFP fusion protein expressed under the control of the
constitutive TEF promoter (Kals et al., 2005). However, it is
possible that this localization does not reflect the normal
situation, because evidence from studies on the transport of
membrane proteins targeted to the inner nuclear membrane
suggests that this fusion protein could not be imported into
the nucleus because the addition of GFP at the C terminus of
Ire1p increases the size of the C-terminal domain beyond
that (?67 kDa) compatible with transport through the lateral
channels of nuclear pore complexes (Holmer and Worman,
2001; Wu et al., 2002).
Membrane-anchored forms of Ire1p in which the majority
of the N-terminal receptor domain of Ire1p is replaced by
bZIP dimerization sequences activate UPR signaling (Liu et
al., 2000), indicating that the Ire1p C-terminal domains of
such constructs are targeted to the intracellular location(s)
required for splicing of HAC1 mRNA. We designed a plas-
mid capable of expressing a membrane-anchored form of a
GFP–Ire1 fusion protein in which the N-terminal receptor
domain is replaced by GFP and an Fv domain, which facil-
itates dimerization upon addition of the cell permeant organic
molecule AP20187 (Spencer et al., 1993; Clackson et al., 1998). In
this construct, which was based upon the pGFP-N-FUS based
vector, DNA sequences encoding the Ire1p ER targeting se-
quence, GFP and Fv were fused upstream of the IRE1 sequence
encoding the transmembrane, linker, kinase, and endoribo-
nuclease domains (Figure 5A). A parallel construct contained
the K646A,R647A substitutions within the NLS in the linker
sequence. These pISGFFVITMCand pISGFFVITMC(K646A,R647A)
plasmids were introduced into RG11907 cells, and after addi-
the fusion proteins were measured by immunoblotting (Figure
5B), whereas their intracellular localization was analyzed by
confocal microscopy (Figure 5C). Confocal images made under
our standard conditions displayed faint perinuclear fluores-
cence in cells expressing the GFFVITMCfusion protein (Figure
5C, first column). This pattern was not apparent in cells ex-
pressing the GFFVITMC(K646A,R647A) protein (Figure 5C, sec-
ond column). Deconvolution of images of cells expressing
GFFVITMC(Figure 5C, third column) revealed a distinct pattern
of perinuclear fluorescence that is very similar to that observed
for a variety of GFP-fused nuclear pore and inner nuclear
membrane proteins localized to the nuclear periphery of yeast
cells (Huh et al., 2003). The fluorescence pattern for GFFVITMC
particularly resembles that reported by Murthi and Hopper
(2005) for Trm1-II, which normally resides as a peripherally
associated protein of the yeast inner nuclear membrane. No
reticular or cortical fluorescence characteristic of ER localiza-
tion (Huh et al., 2003) was observed. Deconvolution of images
of cells expressing GFFVITMC(K646A,R647A) (Figure 5C, fourth
column) confirmed the absence of perinuclear fluorescence,
quences are targeted to the nuclear mem-
brane. (A) Schematic diagrams of i) wild-
type Ire1p and ii) a membrane-anchored
form of a GFP–Ire1 fusion protein in which
the N-terminal luminal domain of Ire1p is
replaced by GFP and Fv. The various do-
mains of the protein are indicated as fol-
lows: S, Ire1p ER targeting sequence; R, N-
terminal receptor domain of Ire1p; TM, Ire1p
transmembrane domain; L, K, E, linker, ki-
nase, and endoribonuclease domains of Ire1p;
GFP, green fluorescent protein; and Fv, vari-
ant FKBP domain. The asterisk in the linker
domain of the GFFVITMCfusion protein indi-
cates the position of amino acid substitutions
in the NLS of the GFFVITMC(K646A,R647A) mu-
tant. (B) RG11907 cells expressing GFFVITMCor
GFFVITMC(K646A,R647A) fusion proteins were
grown at 30°C to an OD600of 0.6 in SC-URA-
MET medium and then treated for 2 h with 2
?M AP20187 dimerizer before cell extracts
were prepared. Equal amounts of total pro-
tein (?200 ?g) were resolved by SDS-PAGE
and analyzed by immunoblotting using anti-
GFP and anti-GAPDH antibodies. (C) Single
colonies of RG11907 yeast cells freshly trans-
formed with plasmids encoding GFFVITMCor
GFFVITMC(K646A,R647A) were grown to an
OD600of 0.6 in SC-URA-MET medium. The cul-
tures were then treated for 2 h with 2 ?M
AP20187 dimerizer before the cells were exam-
ined by fluorescence microscopy. Iterative de-
locity restoration software 3.7.0, and confidence
limit was set to 99.5% or 25 cycles of iteration.
Membrane anchored Ire1p se-
Nuclear Localization Sequence in Ire1p
Vol. 17, December 20065315
indicating that the NLS mutations prevent targeting of the
fusion protein to the nuclear membrane. Instead, a reticular
pattern is observed. Although the “classic” ER pattern includes
brighter perinuclear and cortical fluorescence, the localization
(2003) shows that patterns of generalized reticular fluorescence
consistent with relatively even distribution of the fusion pro-
tein throughout the membrane system of the ER are not un-
usual, particularly for less abundant proteins. We therefore
conclude that the membrane-anchored form of Ire1p is tar-
geted to the inner nuclear membrane by an NLS-dependent
The Basic Sequence in the Ire1p Linker Domain Is
Recognized with High Affinity by Importin ?
To characterize the Ire1p NLS by using in vitro techniques,
we synthesized a peptide containing the cNLS sequence and
the adjacent basic residues for use in an ELISA-based im-
portin binding assay (Hu and Jans, 1999). During synthesis
of this peptide (ENLS-1, residues 642–660 of Ire1p), we also
obtained two N-terminally truncated early termination pep-
tides (ENLS-2; residues 646–660, and ENLS-3; residues 648–
660). The three peptides (see sequences in Figure 6A) were
purified and tested for recognition by the yeast importins
Kap60p/Srp1p (the sole importin ? in S. cerevisiae) and
Kap95p/Rsl1p (importin ?1), which in vivo form the impor-
tin ?/? heterodimer involved in nuclear import of cNLS-
containing substrates (see Figure 6B for a representative
experiment and Table 2 for pooled data).
The Kap60p/Kap95p heterodimer bound ENLS-1 with
very high affinity (1.7 nM). Unexpectedly, Kap95p and
Kap60p alone bound ENLS-1 with affinities (1.6 and 2.2 nM,
respectively) very similar to that of the heterodimer, al-
though Kap60p exhibited somewhat lower maximal bind-
ing. These data imply that ENLS-1 contains recognition sites
for both importin ? and importin ?1. ENLS-2 was also
bound by the heterodimer and by the two individual im-
portins with similarly high affinities (albeit in each case with
reduced Bmax), indicating that the first four amino acids of
the putative cNLS (642KKKR645, absent in ENLS-2) are dis-
pensable for in vitro binding by the importins and suggest-
ing that importin ? may recognize a second minimal cNLS
motif (653KKGR656) present toward the C terminus of both
peptides. ENLS-3 was recognized with greatly reduced af-
finity by both importins, suggesting that the last two basic
amino acids of the putative cNLS (residues K646and R647)
are part of the recognition site for Kap95p and that these
residues may be required (together with653KKGR656) to
form a bipartite cNLS for Kap60p binding.
ELISA was then used to analyze the recognition of the
ENLS peptides by murine importin ?2/Rch1 and its binding
partner importin ?1 (see Figure 6C for a representative
experiment and Table 2 for pooled data). ENLS-1 and
ENLS-2 were both recognized by the importin ?2/?1 het-
erodimer or by importin ?1 with very high affinities similar
to those exhibited by the yeast importins. Again the ENLS-3
peptide bound with low affinity, indicating that residues
K646and R647are part of the minimal ?NLS sequence rec-
ognized by both yeast Kap95p and murine importin ?1, i.e.,
646KRGSRGGKKGRKSRI660. The ENLS-1 peptide was bound
only weakly by importin ?2, indicating that the putative cNLS,
642KKKRKR647, does not represent a high-affinity binding
site for importin ?2, despite its close similarity to the T
antigen cNLS (see above). Importin ?2 is also unable to bind
the alternative cNLS, present within ENLS-2, that is recog-
nized by Kap60p. Thus, the yeast and murine importin ?
proteins differ significantly in their ability to recognize the
portins to synthetic Ire1p NLS peptides as
determined using an ELISA-based binding as-
say. (A) Sequences of synthetic Ire1p NLS
peptides. Lowercase letters represent residues
additional to the Ire1p linker sequence (up-
percase letters). The three peptides were
tested for recognition by yeast importins
(Kap60p/? and Kap95p/?1) (B) or murine
importins (Imp ?2 and ?1) (C). A standard
ELISA-based binding assay was performed
(see Materials and Methods). The KDvalues are
shown on each graph below the peptide
name. Results shown are from a single typical
experiment, performed in triplicate, with
pooled data shown in Table 2.
Binding of yeast and murine im-
L. Goffin et al.
Molecular Biology of the Cell5316
Binding of NLS-bearing cargo to importin ? proteins can
be reversed by RanGTP but not by RanGDP (for review, see
Go ¨rlich and Kutay, 1999). We therefore investigated the
effect of Ran on the interaction of the Ire1p NLS with im-
portin ?1. As shown in Figure 7, RanGTP?S significantly
decreased the binding of ENLS-1 (or a control NLS pep-
tide, PTHrP 67–94) to a GST–importin ?1 fusion protein,
whereas RanGDP consistently had little or no effect on
Kinetic Analysis of Binding of Yeast and Murine
Importins to the Ire1p NLS
We used a BIAcore biosensor to characterize the kinetic
parameters of binding of the yeast and murine importins to
the Ire1p NLS(s). ENLS-1 peptides were linked to the bio-
sensor chip via their N-terminal cysteine residues, and bind-
ing data generated by passing increasing concentrations of
the GST-importin fusion proteins over the immobilized pep-
tides. As shown by ELISA, yeast Kap60p and Kap95p and
murine importin ?1 all showed significant binding, whereas
murine importin ?2 bound ENLS-1 very poorly, if at all
(Figure 8 and Table 2). The affinity of Kap95p for the NLS
peptide (KD? 0.52 nM) was 20- to 25-fold greater than that
of Kap60p (12.5 nM) or murine importin ?1 (11.8 nM). The
association rates of the two yeast importins were compara-
ble (ka? 5.4 and 2.0 ? 104M?1s?1). Murine importin ?1
bound more rapidly (28.3 ? 104M?1s?1) but also dissoci-
ated at an extremely fast rate (330 ? 10?5s?1). Kap95p
displayed the most stable binding, with an apparent disso-
ciation rate (kd? 2.8 ? 10?5s?1), significantly lower than
that displayed by Kap60p (25 ? 10?5s?1). These data sug-
gest that within yeast cells, binding of Ire1p to Kap95p
should be favored over binding to Kap60p and that importin
?-dependent nuclear import should predominate in vivo.
Redundancy of Importin Binding to the Ire1p NLS
To determine whether Kap60p or Kap95p is essential for the
function of Ire1p in UPR signaling in vivo in yeast cells, we
Table 2. Binding parameters for the interaction of importins with synthetic Ire1p NLS peptides
Yeast importin binding parameter
cgKKKRKRG . . . Igy
KRG . . . Igy
G . . . Igy
1.7 ? 0.3 (5)
1.6 ? 0.1 (4)
92 ? 4
2.2 ? 0.5 (4)
66 ? 4
2.5 ? 0.6 79 ? 3.2 3.2 ? 1.1 66 ? 5 5.0 ? 1.1 36 ? 2
83 ? 47 (2)41 ? 13113 ? 26 (2) 30 ? 4 126 ? 40 (3)17 ? 6
Mouse importin binding parameter
Imp?2/?1 Imp?1 Imp?2
cgKKKRKRG . . . Igy
KRG . . . Igy
G . . . Igy
1.7 ? 0.6 (3)
1.9 ? 0.3 (2)
96 ? 0.3
31 ? 20 (2)
81 ? 26
2.7 ? 1.4 (3)101 ? 8.9 3.3 ? 0.2 (2) 83 ? 2182 ? 47 (2) 48 ? 21
45 ? 23 (3) 28.2 ? 6.1 234 ? 60 (2)27 ? 8 558 ? 211 (2) 27 ? 15
ENLS-1 binding parameter
Importin binding parameters were determined using an ELISA-based binding assay as described in Materials and Methods from experimental
data fitted as shown in Figure 6. The results for the apparent dissociation constants (KD, representing the concentration of importin at which
the level of binding is half-maximal) and the maximal level of importin bound (Bmax, normalized relative to that obtained for ENLS-1 when
both ? and ? importins are added) are shown as the means ? SE (n in parentheses), where n is not indicated, the SE is determined from the
curve fit. The apparent association (ka) and dissociation rate constants (kd) derived from the biosensor analysis of the interaction between
immobilized ENLS-1 peptide and importins ?2, ?1, Kap60p, Kap95p, and Kap123p (Figure 8) were calculated from regions of the
sensorgrams where 1:1 Langmurien interactions seemed to be operative. The apparent dissociation constant KDis the concentration of
importin at which the level of binding is half-maximal. The binding of importin ?2 was too low to perform kinetic analysis. The accuracy of
the fit between experimental data and fitted curves (?2) was estimated by chi-square analysis (Catimel et al., 1997; Nice and Catimel, 1999).
Nuclear Localization Sequence in Ire1p
Vol. 17, December 20065317
tested the capacity of yeast cell mutants that are condition-
ally deficient in these importins to support ER stress-medi-
ated HAC1 mRNA splicing. We observed little or no differ-
ence in the extent of stress-induced HAC1 mRNA splicing at
permissive or nonpermissive temperatures in wild-type
yeast cells (strain PSY580) and cells carrying ts mutations in
Kap60p (srp1-31 strain PSY688) or Kap95p (rsl1-4 strain
PSY1103) (Figure 9A, 1-3), even though these strains display
defects in nuclear import in vivo at nonpermissive temper-
atures (Loeb et al., 1995; Koepp et al., 1996). We also analyzed
HAC1 mRNA splicing in a number of other strains bearing
ts mutations or deletions in the genes encoding several
other importin ? family members involved in nuclear im-
port in yeast (Kap104p, Kap108p/Sxm1p, Kap121p/Pse1p,
Kap123p/Yrb4p, or Nmd5p, see Table 1 for strain descrip-
tions and references). Again, no defects in splicing were
observed (Figure 9A; data not shown). The results described
previously (Figure 4) for cells carrying ts mutations in the
genes encoding RanGTPase, RanGAP, and RanGEF indi-
cated that nuclear targeting by the Ire1p NLS involves Ran
cycle-dependent, importin-mediated nuclear transport, sug-
gesting that the lack of a requirement for any individual
importin protein is due to redundant binding of two or more
importins to Ire1p. Because our binding data suggested that
Kap60p would play a minor role in vivo, we surmised that
more than one importin ? family member might recognize
the Ire1p NLS. To test this hypothesis, we first analyzed the
binding of the ENLS-1 peptide to a GST–Kap123 fusion
protein using both the ELISA assay and the BIAcore biosen-
sor. We observed (Figure 9, B and C) that the Kap123 protein
exhibited high affinity for the Ire1p NLS, with binding and
kinetic constants very similar to those previously observed
for Kap95p (Table 2). To determine whether the Ire1p NLS is
recognized by additional members of the importin ? family,
protein extracts were prepared from seven yeast strains each
expressing a different importin ? family member that is
fused at its C terminus to a bright derivative of GFP (More-
house et al., 1999; Seedorf et al., 1999). Their individual levels
of expression were verified by immunoblotting by using the
anti-GFP antibody (Figure 10A). The data shown in Figure
10B demonstrate that all seven of these importin ?–GFP
fusion proteins could be removed from the extracts by in-
cubation with ENLS-4 peptide (see Figure 10 legend for
sequence), which had been linked via its N-terminal cysteine
residues to thiopropyl-Sepharose 6B. The fusion proteins
did not bind to peptide-free resin (see Figure 10B, top row,
second panel for Kap95p-GFP; data not shown for the other
ENLS-1 peptide. Purified recombinant human RanGTP?S and
RanGDP proteins (final concentrations 215 nM) were tested for their
ability to inhibit the binding of ENLS-1 or PTHrP (67–94) peptides
by murine importin ?1 by using an ELISA-based binding assay as
described in Materials and Methods. The sequence of ENLS-1 is
shown in Figure 4A and that of PTHrP (67–94) in Materials and
Methods. The KDvalues derived from data in the absence of added
nucleotides were 2.1 ? 0.58 nM for ENLS-1 and 7.6 ? 3.1 nM for
Effect of Ran proteins on the binding of importin ?1 to
lized ENLS-1 peptide and importins Kap60p, Kap95p, ?2, and ?1.
Varying concentrations (shown on the right of each panel) of yeast
importins Kap60p/? and Kap95p/?1 (A and B) and murine import-
ins ?2 and ?1 (C and D) were injected over immobilized ENLS-1
peptide (see Materials and Methods). The sensorgrams shown have
been corrected for the corresponding signal obtained when the
sample was passed over a blank derivatized control channel.
Biosensor analysis of the interaction between immobi-
L. Goffin et al.
Molecular Biology of the Cell5318
fusion proteins). Addition of nonhydrolysable GTP?S de-
creased the binding of the Kap108 fusion protein to the NLS
peptide-linked resin, presumably by maintaining Ran
present in the cell extract in its active form, whereas the
addition of GDP had no effect (Figure 10C).
A Consensus Sequence Recognized by Multiple Importin ?
The capacity of Ire1p to interact with multiple importin ?
proteins is reminiscent of the interaction of ribosomal pro-
teins and histones with two or more importin ? family
members (Schlenstedt et al., 1997; Rout et al., 1997; Ja ¨kel and
Go ¨rlich, 1998; Claussen et al., 1999; Muhlhausser et al., 2001;
Mosammaparast et al., 2002). Significantly, the combined
NLS we have defined for Ire1p displays a high degree of
similarity to the highly basic sequences present within the ?
importin binding (BIB) domains defined for the yeast L25
and human rpL23a ribosomal proteins and to the short BIB
NLSs defined for Xenopus and human rpL5 and for human
histones H3 and H4 (Table 3A). The NLS of Ire1p also
resembles closely the NLSs defined for eight additional non-
ribosomal proteins known to translocate into the nucleus in
an importin ?-specific manner (Table 3B). Alignment of all
these sequences yielded a “?NLS consensus” sequence (Ta-
ble 3C). When we interrogated the mammalian and yeast
protein sequence databases with this consensus sequence, a
very significant proportion of the proteins identified were
additional ribosomal subunits (see Table 3D). Although
many of the other sequences identified as containing the
consensus were hypothetical ORFs, the set included a num-
ber of nonribosomal proteins confirmed to have a nuclear
localization (Table 3E) as well as a large variety of other
proteins that function in the nucleus, including transcription
factors, polymerases, and mRNA processing enzymes (data
The starting point for this work was a puzzling topological
issue associated with Ire1p, the receptor that senses the load
of unfolded proteins in the ER and transduces the signal
across the ER membrane. The IRE1/ERN1 gene encodes the
first type I transmembrane receptor kinase characterized in
yeast (Mori et al., 1993; Cox et al., 1993) and the first such
receptor in eukaryotic cells known to signal across an inter-
nal membrane. The C-terminal portion of Ire1p carries a
UPR stress-activated endoribonuclease domain that partici-
pates in splicing the mRNA precursor encoding the Hac1p
transcription factor (Sidrauski and Walter, 1997). The ques-
tion arose as to how the cytoplasmic domain of Ire1p could
be targeted to the inner nuclear membrane, the location of
the Rlg1p ligase that completes the unconventional splicing
reaction (Clark and Abelson, 1987). In this study we ana-
lyzed the capacity of a highly basic sequence in the linker
region of Ire1p to function as an NLS both in vivo and in
vitro. We found that the 18 residue basic sequence, which
includes a stretch of six residues that closely resembles a
to the Ire1p NLS. (A) ER stress-induced HAC1
mRNA splicing was assayed in wild-type
yeast (strain PSY580) and strains carrying ts
mutations in SRP1 (Kap60p), RSL1 (Kap95p/
karyopherin ?1), KAP104 (Kap104p/karyo-
pherin ?2), and KAP123 (Kap123p/karyopherin
?4) as described in the legend to Figure 4A. The
relative migration of the spliced (s) and un-
spliced (u) HAC1 mRNA species is indicated on
the right of the panel. (B and C) ENLS-1 and
ENLS-3 peptides were tested for recognition by
yeast importin Kap123p (?4) by using an
ELISA-based binding assay (as described in the
legend to Figure 6) (B) or a biosensor (as de-
scribed in the legend to Figure 8) (C).
Redundancy of importin binding
proteins. (A) Whole cell lysates prepared from PSY580 cells or seven
derivative strains bearing GFP fusions to various importin ? pro-
teins were resolved by SDS-PAGE and immunoblotted by using
?-GFP antibodies (Seedorf et al., 1999). (B) The lysates were incu-
bated with ENLS-4 peptide (NH2-CGKRGSRGGKKGRKSRIGY-
COOH) linked to thiol-Sepharose beads, and the bound fusion
proteins were eluted with DTT. Equal proportions of the total
lysates (T), the unbound fractions (U), and the bound and eluted
fractions (B) were resolved by SDS-PAGE and immunoblotted using
?-GFP antibodies. (C) The ENLS-4/Kap108p interaction is dis-
rupted by a nonhydrolysable GTP analogue. Lysates were incu-
bated in the presence or absence of 1 mM GTP?S or 1 mM GDP
before binding as described in B.
The Ire1p NLS binds to multiple importin ?–GFP fusion
Nuclear Localization Sequence in Ire1p
Vol. 17, December 20065319
Table 3. Amino acid sequence alignment of the Ire1p NLS and the NLSs (defined or putative) of various other importin ?-interacting and
Mammalian importin bindingYeast importin binding
NLS sequence (defined or proposed)
h or y H3(5,6)
KQTARKSTGGKAPRKQL or GKAPRKQLASKAARKSA
h or y H4(5,6)
??1 Tr RBP5 RBP7RBP9
? ??? ????? ?? ?????
??? ????? ??? ?????? ???
? ????? ???
? ????????? ???
KRRQARAKLEAEGKIPK and KERRKYLHESRHRHAMARKR
Membrane protein related to Hsp30 Plasma membrane and nuclear envelope
RNA helicase Nuclear matrix
Cyclophilin PPIaseNuclear matrix, colocalizes with mRNA
Cyclophilin PPIaseSplicing factors
The 19-residue combined Ire1p NLS is shown with the 13-residue minimal importin ?-binding sequence highlighted in bold. A consensus
motif (third box, Z denotes K or R) was defined on the basis of sequence similarities (bolded residues) between the Ire1p NLS and sequences
within either ribosomal and histone proteins (first box) or other proteins (second box) previously reported to interact with one or more
importin ? proteins. Yeast importin proteins are designated ? (Kap60p), ?1 (Kap95p), ?2 (Kap104p), ?3 (Kap121p), ?4 (Kap123p), and ?*
(Kaps 108p, 111p, and 114p). The fourth and fifth boxes contain examples of ribosomal and nonribosomal nuclear proteins identified by
interrogation of mammalian and yeast protein sequence databases as containing an exact or very close fit to the consensus sequence. The
consensus sequence demands only five positions that should be basic residues, but the sequences identified frequently matched the Ire1p NLS
at additional positions (in particular a pair of basic residues aligning with K653 and K654 of Ire1p), providing confidence that we have not
randomly extracted basic sequences. y, yeast; h, human; m, mouse; n, Aspergillus nidulans; r, rat; and x, Xenopus. The references cited are 1.
Schlenstedt et al. (1997); 2. Jäkel and Gärlich (1998); 3. Rosorius et al. (2000); 4. Claussen et al. (1999); 5. Muhlhauser et al. (2001); 6.
Mosammaparast et al. (2002); 7. Chan et al. (1998); 8. Nikolaev et al. (2003); 9. Kahle et al. (2005). The two sequences lie within the portion of
the conserved C-terminal sequence of NF-YA that contains the ncNLS; 10. Xiao et al. (2000); 11. Schedlich et al. (2000). IGFBP denotes
insulin-like growth factor-binding protein; 12. Tiganis et al. (1997). TCPTP denotes T-cell protein tyrosine phosphatase; 13. Lam et al. (1999).
PTHrP denotes parathyroid hormone-related protein; 14. J. R. Aris, annotation of MRH1/YDR033W in the Stanford Saccharomyces Genome
Database; 15. Ursic et al. (1995). The consensus sequence identified lies within the 231 amino acid sequence reported to contain the Sen1p NLS.
16. Bourquin et al. (1997). Human and rat CAR-SCYP are members of the cyclophilin family of peptidylprolyl isomerases. The motif identified
is located outside the cyclophilin domain, in a region not shared with cyclophilin family members that function in other cellular locations.
L. Goffin et al.
Molecular Biology of the Cell 5320
cNLS (Mori et al., 1993), is capable of targeting GFP to the
nucleus of yeast cells in a process that requires proteins
involved in the Ran GTPase cycle that facilitates nuclear
import. The UPR, and in particular stress-induced HAC1
mRNA splicing, is inhibited by point mutations in the Ire1p
NLS that inhibit nuclear localization and also require func-
tional RanGAP and Ran GEF proteins.
Mutagenic analysis and importin binding studies demon-
strated that the Ire1p linker region contains overlapping
potential NLSs: at least one cNLS (within sequences
642KKKRKR647and/or653KKGR656) that can be recognized
efficiently by yeast importin ? (Kap60p), but only poorly by
murine importin ?2, and a novel ?NLS (646KRGSRG-
GKKGRK657) that is recognized by several yeast ? importins
and by murine importin ?1. In vivo in yeast, Ire1p thus has
the capacity to interact with Kap60p or an array of different
importin ?s; hence, it can traffic to the nucleus either via the
classical importin ?/? heterodimer pathway or via various
importin ?-mediated, importin ?-independent pathways.
Clearly, our kinetic data suggest that binding to importin ?
proteins would predominate.
Ja ¨kel and Go ¨rlich (1998) suggested that BIB domains con-
tain an archetypal import signal, still present in many ribo-
somal proteins, which was originally the recognition motif
for the evolutionary progenitor of present importin ?-like
import receptors. They further proposed that during diver-
sification of these receptors in evolution, they acquired ad-
ditional specialized binding sites such as that in importin ?1
for the IBB domain of importin ? or that in transportin for
proteins displaying the M9 NLS. The crystal structure of
importin ?1 (residues 1–485) bound to the nonclassical NLS
of PTHrP (Cingolani et al., 2002) supports this hypothesis by
defining a “prototypical” cargo binding site that is distinct
from the site that interacts with the importin ? IBB domain.
We think that the ?NLS we have identified in Ire1p corre-
sponds to an archetypal BIB motif: it is recognized by at least
seven different importin ? family members and displays
significant sequence similarity to a portion of the BIB do-
mains defined for the yeast L25 and human L23 ribosomal
proteins. The ancient character of this motif seems consistent
with other seemingly archaic features of the UPR signaling
pathway, such as the use of a nonconventional splicing
mechanism (Cox and Walter, 1996; Kawahara et al., 1997),
which shares components of the tRNA maturation system,
to mediate transcriptional control.
Ire1p, which contains an N-terminal hydrophobic signal
sequence for targeting to the ER (Mori et al., 1993), must
initially be inserted across the ER membrane with its N-
terminal receptor domain located in the ER lumen, and its
C-terminal linker, kinase, and endonuclease domains lo-
cated in the cytoplasm. Because the outer nuclear membrane
is contiguous with the rest of the ER membrane system of
the cell, Ire1p could either be co- or posttranslationally in-
serted across the outer nuclear membrane or move to this
location by diffusion from other portions of the ER. Two
possible mechanisms could then be envisaged to transfer
the C-terminal domains of the molecule into the nucleus.
The first model, which has well-characterized precedents
in the activation in mammalian cells of the SREBP receptor
in response to low cholesterol (Brown and Goldstein, 1997)
or of the ATF6 bZIP protein in response to ER stress (Haze
et al., 1999; Yoshida et al., 2000), involves proteolytic cleavage
of Ire1p at or near the cytoplasmic face of the membrane,
followed by import of the released C-terminal domain into
the nucleus. However, we and others have consistently
failed to detect any stress-induced cleavage of Ire1p (Figure
2A; Shamu and Walter, 1996), so we support an alternative
model, which involves movement of the intact Ire1p trans-
membrane protein around the periphery of the nuclear pore,
such that the N-terminal receptor domain remains in the ER
lumen, whereas the C-terminal domains are translocated
into the nuclear matrix. Lateral diffusion of transmembrane
proteins around the pore membrane from the ER membrane
to the inner nuclear membrane of mammalian cells has been
demonstrated for the lamin B receptor and emerin by using
fluorescence recovery after photobleaching (for review, see
Holmer and Worman, 2001). Studies such as these indicate
that the nuclear pore does not pose a barrier to movement of
transmembrane proteins between the outer and inner nu-
clear membranes, provided the nucleocytoplasmic domain
is smaller than ?67 kDa (Holmer and Worman, 2001). How-
ever, this diffusional process is very slow and although
sequences promoting localization to the inner nuclear mem-
brane have been mapped to the nucleocytoplasmic and
transmembrane domains of these and other inner nuclear
membrane proteins (Holmer and Worman, 2001; Wu et al.,
2002), there is no evidence to indicate that the nuclear import
apparatus actively facilitates the transport process. Instead,
localization is apparently driven by trapping of the proteins
in the inner nuclear membrane by their association with
nucleoplasmic components such as lamins and/or hetero-
chromatin, or by interaction between their transmembrane
segments and those of other inner nuclear membrane pro-
teins (Holmer and Worman, 2001; Wu et al., 2002). Our
findings that Ire1p contains an essential importin ?-binding
NLS that targets membrane-anchored GFP–Ire1p to the in-
ner nuclear membrane, together with the very recent report
of importin-mediated inner nuclear membrane localization
of the S. cerevisiae Heh1 and Heh2 proteins (King et al., 2006),
document the interaction of integral membrane proteins
with the nuclear import machinery. This interaction can
apparently occur either via the importin ?/importin ?1
King et al. (2006) to be the sole route for the Heh1 and Heh2
proteins, or in the case of Ire1p, via a variety of different
importin family members. The distinct importin-binding
specificities of the Heh and Ire1 proteins probably reflect
differences in their NLSs; thus, although a 14-residue NLS
(124PKKKRKKRSSKANK137) identified in Heh2p displays
significant sequence identity with the N-terminal “cNLS”
region of the Ire1p NLS (642KKKRKRGSRGGKKGRK657), lit-
tle identity is evident with the extended “?NLS” region
The data reported in this article, as well as our unpub-
lished data on the localization and splicing activity of vari-
ous GFP–Fv–Ire1 fusion proteins (Vodala, S., and Gething,
M.-J., unpublished data) demonstrate a strong correlation
between the degree to which Ire1p is localized in the nucleus
and its capacity to cleave the HAC1 mRNA precursor and
signal the UPR. However, Ruegsegger et al. (2001) reported
that splicing of HAC1 mRNA precursors that have accumu-
lated on stalled polyribosomes can occur in the cytoplasm.
To reconcile these apparently disparate findings, we suggest
that the import of Ire1p into the nucleus is not required for
processing of the preexisting pool of stalled polyribosome-
associated HAC1 mRNA immediately upon induction of ER
stress, but, as the UPR continues, is essential for splicing of
newly synthesized HAC1 mRNA precursor in the nucleus.
Nuclear import of Ire1p may also be necessary to facilitate
its interaction with Ada5p, which is essential for HAC1
mRNA splicing (Welihinda et al., 2000).
Nuclear Localization Sequence in Ire1p
Vol. 17, December 20065321
We thank Clive Slaughter and Brad Reik (University of Texas Southwestern
Medical Center, Dallas, TX) for assistance with peptide synthesis and purifi-
cation, Kazu Mori (Kyoto University, Kyoto, Japan) and Trevor Lithow (Uni-
versity of Melbourne) for gifts of plasmids and antibodies, and Judy Cal-
laghan (University of Melbourne) for advice and assistance with fluorescence
microscopy. This work was supported by a grant to M.-J.G. from the Austra-
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Nuclear Localization Sequence in Ire1p
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