1 The University of Szeged, Faculty of Medicine, Department of Biology, Somogyi
B. u. 4, H-6720 Szeged, Hungary
2 The University of Szeged, Faculty of Medicine, Department of Chemistry,
Szeged, Hungary
3 Department of Organic Chemistry, Eötvös Loránd University,
Budapest, Hungary
4 Biological Research Center of the Hungarian Academy of Sciences, Szeged,
Temesvári krt. 62, H-6720, Hungary
5 Biomedical Research Center, University of Dundee, Level 5, Ninewells Hospital
and Medical School, Dundee, DD1 9SY, UK
* These authors contributed equally to this work
Author for correspondence (e-mail:
szabad{at}comser.szote.u-szeged.hu
)
Accepted 13 January 2002
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Summary |
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Key words: Importin-ß, Nuclear envelope formation, Ran, Dominant-negative mutations, Drosophila
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Introduction |
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In addition to nuclear protein import, importin-ß was shown to have a
role in the organization of microtubules by regulating spatial and temporal
distribution of microtubule-associated proteins throughout the cell cycle
(Wiese et al., 2001;
Nachury et al., 2001
;
Gruss et al., 2001
). Briefly,
during interphase, proteins such as NuMA and TPX2 that are required for
mitotic spindle assembly are retained inside the nucleus. During mitosis,
following the disassembly of the nuclear envelope (NE), the aforementioned
proteins diffuse into the cytoplasm where importin-ß binds and thus keeps
them away from the chromatin. RanGTP was shown to be responsible for releasing
proteins such as NuMA and TPX2, in the vicinity of the chromatin, from
importin-ß or the importin-
/ß heterodimer. Ran is also
involved in NE assembly by attracting and/or binding membrane vesicles
(Zhang and Clarke, 2000
) and
is required for vesicle fusion around chromatin
(Hetzer et al., 2000
).
The association of importin-ß with different types of molecules during
nuclear protein import cycles suggests conformational changes and, in fact,
importin-ß was proposed to be a rather flexible molecule
(Vetter et al., 1999;
Lee et al., 2000
).
Importin-ß is composed from 19 so-called HEAT (armadillo) repeats
arranged in a Spanish collar type of superhelix. It binds (1) Ran at its
N-terminal region; (2) the nucleoporins with a large middle section; and (3)
the importin-ß binding (IBB) domain of importin-
with its
C-terminal region (Kutay et al.,
1997
; Wozniak et al.,
1998
). In fact, the spatial structures of importin-ß, on its
own (Lee et al., 2000
) and in
complex with RanGTP (Vetter et al.,
1999
), as well as that of the IBB domain of importin-
(Cingolani et al., 1999
) have
recently been elucidated.
Although the KetelD eggs, deposited by the
KetelD/+ females, appear normal and are fertilized,
cleavage nuclei do not form inside. The KetelD egg
cytoplasm is very toxic: when injected into wild-type cleavage embryos it
hinders formation of cleavage nuclei
(Tirián et al., 2000).
Surprisingly, however, when injected into wild-type cleavage embryos the
KetelD egg cytoplasm does not prevent nuclear protein
import. Moreover, nuclear proteins are imported into nuclei of
digitoninpermeabilized HeLa cells in the presence of ovary extracts of the
KetelD/+ females
(Lippai et al., 2000
); thus
revealing novel importin-ß function.
In the present report we show that the replacement of Pro446 by Leu in three of the four independently isolated KetelD alleles dramatically changes the function of importin-ß. We propose (based on CD spectroscopy of model peptides) that the flexibility of importin-ß is reduced upon replacement of Pro446 by Leu. We show that the P446L mutant protein has no detectable affinity to RanGTP but binds RanGDP. We present evidence that the KetelD-encoded protein does not prevent nuclear protein import carried out by normal importin-ß but inhibits NE formation at the end of mitosis. We also report that replacement of Ser317 by Thr restores characteristic importin-ß functions: Drosophila females that lack a functional Ketel gene but carry a transgene with both the S317T and the P446L mutations are fully viable and fertile.
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Materials and Methods |
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Molecular cloning and sequencing of KetelD1, one
of the KetelD alleles
DNA of KetelD1/ larvae served as the template in
a set of PCR reactions to produce DNA fragments for sequencing. The PCR
primers were designed based on the Ketel genomic sequence available
in the EMBL nucleotide sequence database under the accession no. AJ002729.
Sequencing was carried out by using the dideoxy method in an IBI automated
sequenator on both strands. Results of sequencing were compared with the
sequence reported for the wild-type allele
(Lippai et al., 2000).
The KetelD (KD)
transgenes
The presumptive KetelD1 mutation was generated through
in vitro mutagenesis in PCR reactions. The generated
BglII-ClaI fragment replaced the corresponding DNA segment
in a plasmid containing a 4.0 kb Xba-BamHI genomic fragment, which
includes the Ketel promoter and the 5' segment of the
Ketel coding region, combined with a 2.3 kb cDNA fragment
representing the rest of the transcribed part of the Ketel gene [see
figure 1b in Lippai et al.
(Lippai et al., 2000)]. The
above sequences were cloned into the CaSpeR vector with the
mini-white reporter gene and germ line transformants were generated
by standard procedures (Thummel et al.,
1988
). Flies carrying the KD transgene have
orange eyes of different intensities on white genetic background.
Three independent KD transgenic lines were recovered. The
KD transgenes are inserted into different positions of
third chromosomes and are maintained as the third chromosome-linked
Fs mutations (Erdélyi and
Szabad, 1989
). The expression level of the KD
transgenes was characterized by measuring eye pigment content of w/w;
KD/+ flies. Eye pigment contents were determined by extraction
of eye pigments and photometry as described by Reuter and Wolff
(Reuter and Wolff, 1981
). The
KD transgenes were used for the generation of +/+;
KD females and ketelnull/;
KD zygotes.
|
For production of transgenes with both the S317T and the P446L mutations, we replaced a BglII-BstEII fragment in the vector containing the KD transgene with the same fragment containing the S317T mutation coding DNA fragment.
Production of the KetelD-encoded protein in
bacteria
We produced normal Ketel protein as described
(Lippai et al., 2000). For
production of the P446L mutant protein in E. coli, an expression
vector containing full length Ketel cDNA with the P446L
mutation was generated by replacing the BglII-ClaI section
in the pQE30 expression vector that contains the wild-type Ketel
cDNA. Construction of the pQE30 expression vector with the wild-type
Ketel cDNA was described previously
(Lippai et al., 2000
).
The expressed proteins included an N-terminal His tag that allowed purification on a nickel-NTA agarose column. Unlike the normal Ketel protein, over 95% of the P446L mutant protein were present in inclusion bodies. To produce functional P446L mutant protein, we dissolved the inclusion bodies in 6 M guanidine hydrochloride in the TNM buffer (50 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl2 and 5% glycerol) as used during purification of the normal Ketel protein. Renaturation of the P446L mutant Ketel protein was achieved through a 6 to 0 M decreasing guanidine gradient in TNM buffer and eluted with 0 to 0.5 M imidazole gradient in TNM buffer. The eluted protein was dialyzed against 0.1 x TNM buffer overnight. Two hundred µl aliquots of the dialyzed protein were lyophilized and stored at -70°C. When used, the aliquots were dissolved in 20 µl H2O. To test biological activities of the purified P446L mutant Ketel protein we (1) injected it into wild-type, histone-GFP and lamin-GFP-expressing cleavage embryos; (2) used it in the nuclear import assay system with digitonin-permeabilized HeLa cells; and (3) used it in solutions to study Ketel-Ran interactions.
Injections of P446L protein into cleavage embryos
To visualize effects of the P446L molecules on cleavage embryos we carried
out the following injection experiments. (1) We injected into wild-type
embryos approximately 200 picolitres/egg (2% total egg volume) from a
solution that contained wild-type or P446L mutant importin-ß protein (1.2
µM) and a fluorescent import substrate (0.24 µM). The fluorescent
substrate was a pentamer of a fusion protein in which the nucleoplasmin core
domain was combined with the importin-ß-binding domain from
importin-
[IBB core pentamer
(Lippai et al., 2000
)]. (2)
The wild-type or P446L mutant importin-ß solutions were injected into
embryos in which the chromatin was labeled by histone-GFP protein
(Clarkson and Saint, 1999
). (3)
The wild-type or the P446L mutant importin-ß protein solutions were
co-injected with a 1% solution of the 170 kDa red fluorescent TRITC
(tertramethylrhodamine isothiocyanate isomer R)-dextrane. (4) Wild-type or
P446L mutant importin-ß was injected into cleavage embryos in which a
UAS-tubulin-GFP construct (Grieder et al.,
2000
) was driven by a nanos-Gal4 driver. (5) The wild-type or
P446L mutant importin-ß solution (1.2 µM) was also injected into
cleavage embryos in which a UAS-lamin-GFP construct was driven by a
nanos-GAL4-VP16 driver (Van Doren et al.,
1998
). (The UAS-lamin-GFP transgene was kindly provided by N.
Stuurman; see the FlyBase website
http://flybase.bio.indiana.edu
). Following injections, the fate of the injected embryos was followed through
optical sections in a Zeiss LSM410 confocal microscope. The injections were
done at 20°C.
The nuclear protein import assay
Digitonin-permeabilized HeLa cells were prepared by a modified protocol
(Adam et al., 1990). Briefly,
HeLa cells were grown on coverslips to 50-80% confluence, washed in ice-cold
permeabilization buffer (20 mM Hepes-KOH pH 7.5, 110 mM potassium acetate, 5
mM magnesium acetate, 250 mM sucrose and 0.5 mM EGTA) and permeabilized for 15
minutes in the same buffer containing 60 µg/ml digitonin. The coverslips
were washed three times in permeabilization buffer without digitonin.
Coverslips were incubated with each 20 µl of import reaction. The import
buffer contained 2 mg/ml nucleoplasmin core (to block nonspecific binding), 20
mM Hepes/KOH pH 7.5, 140 mM potassium acetate, 5 mM magnesium acetate, 250 mM
sucrose, 0.5 mM EGTA. Where indicated, reactions were supplemented with an
energy regenerating system (0.5 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate,
50 µg/ml creatine kinase) and Ran mix [3 µM Ran-GDP, 150 nM RanGAP
(Rna1p from yeast), 300 nM NTF2 and 150 nM RanBP1]. Nuclear import of the IBB
core pentamer was monitored in optical sections. Import reaction samples
contained 0.24 µM fluorescein-labeled IBB core pentamer, 1.2 µM
wild-type or P446L mutant Ketel protein and, where indicated, Ran mix and an
energy regenerating system. Reactions were stopped after 5 minutes by fixation
in 3% paraformaldehyde (w/v) in PBS, washed in PBS and water, and mounted with
2 µl of vectorshield mounting medium (Vector).
Binding assays and immunoprecipitations
Binding assays were carried out as described
(Hughes et al., 1998).
Briefly, GST-Ran loaded with GDP or GST-RanQ69L (a RanGTP frozen form of Ran)
loaded with GTP were incubated with wild-type and KetelD
egg extracts as well as wild-type and P446L mutant Ketel proteins expressed in
E. coli and purified as described in Lippai et al.
(Lippai et al., 2000
) and in
this paper. Following binding Ran was pulled down by glutathione-Sepharose
beads. The beads were recovered and washed, and the bound proteins were
separated by SDS-PAGE and immunostained using affinity-purified anti-Ketel
antibody (Lippai et al.,
2000
). The anti-Ketel antibody is equally efficient in recognizing
the wild-type and the P446L Ketel proteins. The concentration of wild-type and
P446L Ketel proteins was 0.3 µM. Protein concentrations of the egg extracts
were adjusted to 18 mg/ml.
For immunoprecipitations we incubated Protein-A-agarose beads with the polyclonal anti-Ketel antibody and after through washing the anti-Ketel beads were given to wild-type or KetelD egg extracts; when indicated an energy regenerating system (0.5 mM ATP, 0.5 mM GTP, 20 mM creatine phosphate and 100 µg/ml creatine kinase) and 3 µM of wild-type or P446L Ketel proteins were added. The precipitated Ran was detected by western blot.
Enzymatic assays
Labeling of Ran with [-32P]GTP and GTPase assays were
performed essentially as described
(Görlich et al., 1996
).
Concentration of the proteins were as follows: Ran 0.3 µM, wild-type and
P446L Ketel 1 µM, RanBP1 0.4 µM, Drosophila RanGAP 25 nM.
Hydrolysis of Ran[
-32P]GTP to RanGDP and the release of
[
-32P] was measured in a liquid scintillation counter 2
minutes after bringing the components together. For measuring of nucleotide
exchange activity of RCC1 on Ran, human Ran protein was loaded with
[3H]GTP or [3H]GDP. Protein concentrations in the
reactions were as follows: 0.3 µM Ran, 1 µM wild-type and P446L Ketel
and 30 nM RCC1. The exchange of labeled GTP or GDP to unlabeled GDP was
measured for 2, 3 and 4 minutes in a liquid scintillation counter after the
components were brought together.
Structural analysis
As shown in Table 3, we
synthesized two model polypeptides. (1) The first included helix B of HEAT
repeat 10, the linker region plus helix A of the HEAT repeat 11
(Cingolani et al., 1999). The
linker region, like the wild-type Ketel protein in the 446th position (441st
in human importin-ß), contained a proline
(Table 3). (2) The second
polypeptide differed from the first by one amino acid: the proline in the
linker region was replaced by leucine.
|
The polypeptides were synthesized by standard solid phase technique using
Boc (butyl-oxy-carbonyl) chemistry and an automated ABI 430A synthesizer
(Merrifield, 1963). The crude
peptides were purified by reverse-phase HPLC and characterized by mass
spectrometry. The polypeptides were dissolved in (1) 100% trifluoro-ethanol
(TFE); (2) a mixture of 67% TFE and 33% H2O; and (3) 33% TFE and
67% H2O. CD spectra of the solutions were recorded on a Jobin Yvon
dichrograph Mark VI in a 0.02 cm cell. The concentration of the samples varied
between 0.1-0.5 mg/ml. CD spectra were analyzed using a Convex Constrain
Analysis Plus software.
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Results |
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The CTC mutant sequence is part of the CTCGAG palindrome that is in fact an
XhoI restriction site and hence the
KetelD1-resulting C4114T transition created a new
XhoI restriction site. We isolated DNA from
KetelD2/, KetelD3/ and
KetelD4/ hemizygous larvae, PCR amplified the
region around the XhoI site and subjected the DNA to XhoI
digestion. (As control, DNA was also isolated and XhoI-digested from
flies that carried the founder chromosomes on which the
KetelD alleles were induced.) Results of XhoI
digestion clearly revealed the presence of a new XhoI restriction
site in the KetelD3 and in the KetelD4
but not in the KetelD2 allele. (Data not shown.)
Apparently a common C4114
T transition is associated with three of the
four independently isolated KetelD mutations and all three
lead to replacement of Pro446 by Leu.
The KetelD (KD) transgenes
act as the KetelD mutations
To determine whether the C4114T transition did indeed lead to
formation of three of the four KetelD alleles, we
generated three KD transgenic lines: A, B and C, with the
in vitro generated C4114
T transition inside. The KD
transgene became inserted into different sites on different third chromosomes.
Three features of the KD transgenes confirm that the
KetelD-related phenotypes are consequences of the
C4114
T transition in the Ketel gene.
The KD transgenes completely or largely sterilize
females
The +/+; KD females (with two normal Ketel
genes and one KD transgene) are either sterile or their
fertility is severely reduced (Table
1). Whereas the `C' KD transgenic line renders
females completely sterile, as was described for the
KetelD1/+/+ females which, in addition to
KetelD1, carried two normal (+) Ketel gene copies
(Tirián et al., 2000),
very low offspring production rates are characteristic for lines A and B
(Table 1). The variability in
the female-sterilizing ability of the three KD transgenic
lines can be best explained by the different expression levels of the
transgenes. Since the mini-white marker gene is also included in the
KD transgenes, eye pigment content of the transgenic flies
is the measure of the expression level of the transgenes
(Table 1). While eyes of the
line `C' flies are dark orange and reveal intensive expression of the
KD transgene, eyes of the line `A' flies are light orange
and reflect a low expression level. However, even the low expression level
renders the +/+; line `A' KD females almost completely
sterile (Table 1).
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Production of offspring by the +/+; KD line `A' females provided an opportunity to recover a few +/+; KD/KD females. As expected, they had dark orange eyes and were completely sterile and, as in case of the KetelD1/+ females, embryogenesis did not commence in their eggs.
The KD transgenes, like the
KetelD alleles, act as dominant-negative mutations
The KetelD mutations are of dominant-negative type
(i.e. their female sterilizing effect can be slightly reduced by extra doses
of wild-type Ketel alleles)
(Tirián et al., 2000).
To determine whether the mutation in the KD transgene
possesses dominant-negative features, we combined the line `A'
KD transgene with three different K+
transgene lines (`N', `J' and `K'; Table
2), which carry a normal Ketel gene inside
(Lippai et al., 2000
). Results
of the experiment are summarized in Table
2 and clearly show the dominant-negative nature of the mutation in
the KD transgene: offspring production of the +/+;
KD; K+ females significantly exceeded
those of the +/+; KD ones (P<0.01;
2 test). The difference in the
KD-compensating effect of the K+
transgenic lines correlates well the with expression level of the
mini-white reporter gene: the more intensively the
K+ transgene is expressed, the more eye pigment flies have
and the more effectively the K+ transgene reduces
KD-imposed female sterility
(Table 2).
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The KD transgenes do not support zygotic
development
Efforts to construct ketelnull/-; KD flies,
that lack functional Ketel gene and carry one of the three
KD transgenes, failed: not a single
ketelnull/-; KD adult emerged among the well
over 1000 offspring recovered in the case of each of the three
KD transgenic lines. The ketelnull/-;
KD zygotes, like the ketelnull/- and the
KetelD/- ones die during second larval instar. Unlike the
KD transgenes the K+ transgenes, with
normal Ketel gene in the transgene, completely rescue lethality
associated with loss of Ketel gene function: the
ketelnull/-; K+ flies are fully viable
and fertile (Lippai et al.,
2000; Tirián et al.,
2000
). Apparently the P446L mutation in the
KD transgenes behaves as the KetelD
alleles: when paternally derived it acts as the ketelnull
recessive zygotic lethal mutations.
The P446L mutant Ketel protein inhibits formation of intact NE when
injected into wild-type cleavage embryos
Cytoplasm of the KetelD eggs is exceedingly toxic: when
injected into wild-type cleavage embryos the KetelD egg
cytoplasm prevents formation of nuclei at the end of mitosis
(Tirián et al., 2000).
To determine whether the P446L importin-ß molecules (produced and
purified from E. coli cells) possess the same effect as the
KetelD egg cytoplasm, we injected small volumes of the
P446L protein solution along with a fluorescent nuclear substrate
into wild-type cleavage Drosophila embryos. As illustrated in
Fig. 1A and D, the nuclear
substrate entered the cleavage nuclei irrespectively of whether wild-type or
P446L mutant importin-ß protein solutions were injected. It is important
to note that the P446L protein did not disrupt the NE. Similarly, ovary
extracts of the KetelD/+ females did not block
accumulation of the fluorescently labeled import substrates into digitonin
permeabilized HeLa cells, and nuclei remained intact for at least 4 hours in
the presence of mutant P446L protein
(Lippai et al., 2000
). During
the upcoming mitosis the fluorescent substrate was homogeneously distributed
in the egg cytoplasm around the site of injection, which indicated disassembly
[that is partial in Drosophila
(Foe et al., 1993
)] of the NE
and the concomitant release of the fluorescent substrate into the egg
cytoplasm (Fig. 1B,E).
Following termination of mitosis, in the control the fluorescent substrate
entered the newly forming nuclei that doubled in number
(Fig. 1C). In the P446L mutant
protein, however, the fluorescent substrate remained homogeneously distributed
at the site of injection (Fig.
1F). Should intact NE form, the high molecular weight fluorescent
substrate would be either excluded (in the absence of nuclear protein import)
from the newly forming nucleus leaving a dark outline of the nucleus, or
re-imported (where nuclear protein import has resumed) in which case the
nuclei would be highlighted by the fluorescent substrate, as seen in the
control experiment (Fig. 1C).
Since neither of the expected versions occurred, it appears that the P446L
Ketel protein prevents formation of intact cleavage nuclei. Evidently, the
P446L mutant importin-ß exerts the same toxic effect on wild-type
cleavage embryos as the KetelD egg cytoplasm and is very
efficient in abolishing the function of wild-type importin-ß
molecules.
One possible explanation for the failure of cleavage nuclei formation is that the P446L molecules induced decay of the chromatin around which the NE would have assembled. To clarify the `chromatin decay' possibility, we injected purified P446L mutant importin-ß into the posterior end of cleavage embryos that expressed GFP-tagged histone highlighting the chromatin. The fate of the chromatin was followed through two rounds of cleavage divisions in the injected embryos. As time lapse recordings revealed, the P446L molecules did not hinder the upcoming chromosome segregation (Fig. 2D-F), which proceeded in almost the same way as chromosome segregation in the anterior part of the embryo that was free of P446L protein (Fig. 2A-C). While chromatin highlighted by histone-GFP persists at the posterior end of the embryo and forms aggregates, further cleavage cycles are accomplished at the anterior end (data not shown). Results of the histone-GFP experiments ruled out the possible decay of chromatin as the reason for the failure of cleavage nuclei formation in the presence of P446L mutant importin-ß. Although chromosomes always segregated, the distance between the chromatin blocks did not grow subsequently, leading to the formation of structures that appeared as chromatin aggregates. To examine whether the formation of chromatin aggregates is the consequence of persisting or abnormally organized mitotic spindles, we injected wild-type and P446L mutant importin-ß into tubulin-GFP expressing Drosophila cleavage embryos and monitored behavior of the mitotic spindles. As Fig. 3A-D shows, the injection of wild-type importin-ß has no affect on mitotic spindle formation, shape and disassembly. Following the injection of P446L importin-ß (Fig. 3E-H) mitotic spindles form normally and the spindle elongation and disassembly is not affected. However, the homogenous distribution of the GFP-tubulin (Fig. 3H) indicates that NE failed to assemble since GFP-tubulin is not excluded from the space where nuclei should have formed. Failure of the chromosomes to move apart during interphase is most likely the consequence of the failure of NE formation.
|
|
To elucidate the possibility of failure of NE assembly, we conduced two further sets of injection experiments. To test whether or not intact NE forms around the chromatin in the presence of P446L protein, we co-injected a high molecular weight red-fluorescent dextrane with the P446L protein into histone-GFP expressing cleavage embryos. If intact NE is assembled around the chromatin the dextrane is expected to be excluded from the nuclei. If, however, functional NE fails to form around the chromatin, the dextrane is expected to possess an almost homogenous distribution. Following co-injection of wild-type importin-ß and the red-fluorescent dextrane, the red and the green (chromatin-derived) signals were clearly separated: the green signal originated from the inside of the interphase cleavage nuclei and the red signal from the cytoplasm, which shows the formation of cleavage nuclei and hence functional NE (Fig. 4A,B). In the case of P446L and red-fluorescent dextrane co-injections the dextrane-derived signal was basically homogeneously distributed (Fig. 4D) even though the histone-GFP highlighted chromatin resembled normal interphase chromatin (compare Fig. 4A,C). Results of the above experiment are in agreement with the failure of functional NE formation in the presence of the P446L mutant importin-ß.
|
To visualize the effect of the P446L-protein-induced NE defect, we injected
wild-type (as control) or P446L mutant importin-ß solutions into cleavage
embryos in which lamin-GFP highlighted the lamin lining of the internal NE
surface. Most of the lamin is phosphorylated upon entry into mitosis and the
residual lamin-GFP molecules faintly show the so-called spindle envelope.
(Cleavage mitosis in Drosophila is an intermediate between closed and
open mitosis.) Upon entering the upcoming mitosis the lamin molecules re-enter
the nucleus and highlight the NE. It is to be expected that if the P446L
mutant molecules prevent NE assembly there will be no lamin-GFP signal
outlining the NE at the site of injection. As
Fig. 5A-C shows, following the
injection of wild-type importin-ß the lamin-GFP molecules highlight the
NE during the upcoming interphase. Upon entry to mitosis, most lamin molecules
diffuse into the cytoplasm and only some remain attached to the spindle
envelope (Paddy et al., 1996)
(Fig. 5B). Following chromosome
segregation the NE reassemble as pictured by formation of the green
fluorescent lamin lining (Fig.
5C). When the P446L mutant protein is injected into the
lamin-GFP-expressing cleavage embryos, the lamin disappears during mitosis as
in the control experiment showing that mitosis is not affected until late
anaphase (Fig. 5D-F). In the
presence of the P446L molecules, however, the lamin lining never re-forms,
which shows the failure of intact NE assembly.
|
The above injection experiments show that the P446L importin-ß
molecules interfere with the formation of intact cleavage nuclei and the
defect is manifested during the mitosis-to-interphase transition through the
prevention of intact NE assembly. The KetelD mutations
possess dominant-negative action on NE assembly and impede function of the
normal importin-ß molecules and thus reveal a novel role of
importin-ß required during NE formation at the end of mitosis. The novel
importin-ß function is distinct from both its role in nuclear protein
import and the recently described function in mitotic spindle assembly
(Wiese et al., 2001;
Nachury et al., 2001
;
Gruss et al., 2001
).
The P446L mutant importin-ß allows formation and docking on the
NE of the import complexes but does not support nuclear protein import
As described earlier, the wild-type importin-ß molecules support
import of NLS-containing substrates into nuclei of digitonin-permeabilized
HeLa cells (Lippai et al.,
2000). Interestingly, ovary extracts of the
KetelD/+ females, with both wild-type and P446L mutant
protein inside, support nuclear protein import as efficiently as ovary
extracts of wild-type (+/+) females
(Lippai et al., 2000
). As
presented above, when injected into wild-type embryos the P446L mutant protein
does not prevent import of a nuclear substrate into the cleavage nuclei. Two
feasible possibilities seem to account for the above phenomena: (1) it may be
that, although the P446L mutant protein molecules do not participate in
nuclear protein import, they do not prevent function of the wild-type
importin-ß molecules to accomplish their function; or (2) perhaps P446L
mutant importin-ß supports nuclear protein import. To determine which of
these two possibilities is true we analyzed behavior of the P446L mutant
protein in the nuclear protein import assay. As illustrated in
Fig. 6, in the presence of only
the fluorescent nuclear substrate and importin-ß or the P446L mutant
protein, although in reduced amounts, nuclear import complexes form and dock
on the cytoplasmic surface of digitonin-permeabilized HeLa cell nuclei
(Fig. 6A,C). The higher
cytoplasmic background, in the case of P446L, is most likely the consequence
of the altered structure of the P446L molecules (see below) leading to
association of the import cargo/P446L importin-ß with cytoplasmic
structures, possibly membranes or microtubules. In the presence of normal
importin-ß and when further components of nuclear import are added (i.e.
Ran, NTF2, RanGAP, RanBP1 and energy supply) the complexes are imported into
the nuclei (Fig. 6B). In the
case of the P446L mutant protein, however, import complexes do not form
(Fig. 6D) as revealed by the
absence of fluorescent signal in the HeLa cells.
|
Results of the above experiments show that the P446L mutant protein does
not support nuclear protein import. However, as the above-mentioned injection
experiments and nuclear import assays with ovary extracts of
KetelD/+ females revealed
(Lippai et al., 2000;
Tirián et al., 2000
),
they do not hinder nuclear import accomplished by the normal importin-ß
molecules. Apparently effects of the P446L mutant protein are manifested only
during the mitosis-to-interphase transition in preventing intact NE
formation.
The P446L mutant protein loses affinity to RanGTP but binds
RanGDP
Changes in the Ran-binding ability of the P446L protein are suggested by
the fact that the P446L molecules are unable to accomplish nuclear protein
import in the digitoninpermeabilized HeLa cells
(Fig. 6D). To examine this
possibility, we tested the binding of wild-type and P446L importin-ß to
different GST-Ran fusion proteins in solution binding assays
(Fig. 7A). Apparently, RanGDP
binds significantly higher amounts of importin-ß rom
KetelD egg extracts than that from wild-type
Drosophila egg extracts. Since Ran is mainly in its GDP-bound form in
cytoplasmic extracts, the above result suggests increased RanGDP binding
affinity of the P446L protein. Conversely, RanQ69L loaded with GTP binds
higher amounts of importin-ß from wild-type egg extracts than that from
KetelD extracts, suggesting reduced binding ability of
P446L to RanGTP. As a negative control we used GST protein alone, which showed
only background binding levels with both P446L and wild-type importin-ß.
Since the KetelD egg extracts contain 50% wild-type
importin-ß, the extracts are not suitable to examine the RanGTP binding
ability of the P446L protein. To confirm the reduced affinity of P446L to
RanGTP, we measured the amount of the pulled down importin-ß proteins
from solutions containing purified wild-type or P446L mutant importin-ß.
As shown in Fig. 7A, wild-type
importin-ß binds strongly to RanQ69L, but the P446L protein shows only
background binding to RanQ69L. (RanQ69L is a GTP-loaded GTPase deficient
mutant Ran protein.)
|
To support the altered binding of P446L to Ran, we carried out immunoprecipitations with the polyclonal anti-Ketel antibody. The amount of precipitated endogenous Ran was higher from KetelD egg extracts compared with wild-type egg extracts. However, if an energy regenerating system and purified wild-type or P446L importin-ß is added to the wild-type extract, more Ran is precipitated from the extract supplemented with the wild-type importin-ß (Fig. 7B). The shift in Ran binding ability following addition of an energy supply correlates with the ability of wild-type importin-ß to bind RanGTP and the inability of P446L to do so. Results of the described experiments are further supported by the enzyme assays described below.
Importin-ß has been known to inhibit both GTP hydrolysis on Ran and the exchange of RanGTP catalyzed by RCC1. We studied, in solutions, the effects of the purified importin-ß and P446L proteins on both nucleotide exchange and GTP hydrolysis on Ran. The wild-type Ketel protein inhibits both GTP nucleotide exchange and GTP hydrolysis, whereas the KetelD encoded protein has no effect on the processes (Fig. 7C,D), which shows that the P446L mutant protein cannot bind to RanGTP. Neither wild-type nor P446L have significant effect on nucleotide exchange from RanGDP (data not shown). In conclusion, the failure of functional NE formation may be the consequence of the altered RanGTP binding ability of the P446L mutant importin-ß.
The P446L mutation appears to increase helix content and
reduce flexibility of the encoded protein
As described above, the C4114T transition leads to replacement of a
helix-breaking Pro by Leu in position 446. It may be that in the P446L mutant
protein the Pro446
Leu exchange leads to fusion of two adjacent helices,
namely the B helix of HEAT repeat 10 and the short helix in the linker region
towards helix A of HEAT repeat 11. To test whether helix content of the P446L
molecules is indeed increased compared with the wild-type importin-ß
molecules, we synthesized two model peptides representing the noteworthy
region in importin-ß. We then carried out CD spectroscopy of the peptides
that were dissolved in the apolar solvent trifluoro-ethanol (TFE) or in
mixtures of TFE and water (Fig.
8). CD spectroscopy has been known to be a sensitive technique to
analyze protein structures: CD spectra recorded in different polarity
molecular environments (i.e. in solvents with different dielectric constant)
often reveals structural changes (Perczel
et al., 1991
; Perczel et al.,
1992
).
|
In 100% TFE, which provides low polarity and a membrane mimicking
environment, both model peptides resulted in so-called C-type CD spectra,
which refers to a mixture of ß-turns (type I or III) and - (or
310) helices (Perczel et al.,
1991
). Ordered structure of the peptides is revealed by (1) the
presence of the two shoulders in the spectra at 209 and 224 nm (the
* and n
* transitions, respectively) and also
by (2) reaching the 209 nm shoulder lower
values as the 224 nm shoulder
(Fig. 8). CD spectra of the
Pro-containing peptide representative of the wild-type importin-ß
reveal high
-helix content in both TFE and mixtures of TFE and
water and the spectra changed significantly along with increase in water
content of the molecular environment, reflecting flexibility of the peptide
(Fig. 8A). Results of CD
spectroscopy are in line with the previously published 3D structure of the
aforementioned segment of importin-ß
(Cingolani et al., 1999
;
Lee et al., 2000
) and are
illustrated in Fig. 9B: amino
acids that comprise HEAT repeats in 10B and 11A form
-helices and are
interconnected with a ß-turn. There is a short, three amino acid
-helix starting with Pro446 in the turn region adjacent to HEAT repeat
10B.
|
In contrast to the Pro-containing peptide, CD spectra of the Leu-containing
model peptide, representing the P446L mutant protein, remained essentially
unchanged on increasing the water content (i.e. the dielectric value) of the
medium, which shows that the Leu-containing peptide, while keeping its
predominantly helical organization, loses flexibility compared with the
Pro-containing peptide (Fig.
8B). It appears that in the presence of water, helices of the
Pro-containing peptide undergo a limited untwisting and/or rearrangement of
the helix-turn-helix conformation and the ProLeu exchange results in a
mutant molecule that lost flexibility. An illustration of the altered
molecular structure is shown in Fig.
9D, considering that the Pro
Leu exchange increased
-helix content of the peptide.
Computer modeling of the importin-ß and the P446L mutant
importin-ß proteins
Based on results of the formerly described CD spectroscopy, we carried out
computer 3D modeling of both the normal and the P446L mutant importin-ßs.
We took X-ray crystallographic data of human importin-ß associated with
the IBB domain of importin-. [For atomic coordinates of importin-ß
see access code 1QGK (Cingolani et al.,
1999
).] We assume that the human importin-ß 3D data used for
the modeling is adequate because: (1) the size of human and
Drosophila importin-ßs are similar: 876 and 884 amino acids; (2)
amino acid sequences of human and Drosophila importin-ßs share
60% amino acid identity and 78% similarity
(Lippai et al., 2000
); and (3)
The 10B HEAT repeatlinker11A HEAT repeat region is evolutionary
highly conserved: the amino acid identity and similarity are 64.1% and 92.3%
(Table 3). Pro446 resides in
the linker region between the B helix of HEAT repeat 10 and the A helix of
HEAT repeat 11 at the beginning of a small 3-amino-acid-long
-helix
after HEAT repeat 10 (Fig. 9)
(Cingolani et al., 1999
). CD
spectra of model peptides representing the wild-type and the P446L mutant
proteins reveal loss of flexibility upon Pro446
Leu replacement and the
lost flexibility is most likely the consequence of the B helix of HEAT repeat
10 fusing with the short
helix in the linker region. Changing the
and
angles of Leu439 and Leu440 such that the two amino acids fit
into a fused
-helix, an open molecular conformation emerges
(Fig. 9). As a consequence of
opening the Spanish collar, the hydrophobic internal surface of the molecule
becomes exposed to water. The consequence of the amino acid exchange may be
less dramatic in the whole molecule due to its flexibility, but a significant
conformational change is supported by the more hydrophobic behavior of the
P446L mutant protein (i.e. it was much more difficult to express and purify
compared with normal importin-ß), and by the fact that the exchange of a
distantly located residue suppresses the mutant defects caused by the P446L
protein (see below).
S317T is an intragenic dominant suppressor mutation of
P446L and restores Ketel gene function
Following molecular analysis of the ketelr alleles,
which are revertant alleles of the KetelD mutations and
were induced through second mutagenesis
(Szabad et al., 1989;
Erdélyi et al., 1997
),
we identified S317T, which is an intragenic dominant suppressor
mutation of P446L. The S317T mutation originated through a
single T
A transversion in the 3656th position and led to replacement of
Ser317 by threonine. We generated five transgenic lines (labeled
KS317T&P446L), which included both the S317T
and the P446L mutations. The +/+;
KS317T&P446L females with two wild-type Ketel
alleles (+) and with the KS317T&P446L
transgene are fully fertile, which suggests that the S317T mutation
annulled the P446L-imposed dominant female sterility. That the
KS317T&P446L transgene does indeed restore
Ketel gene function is best shown by the fact that the
ketelnull/-; KS317T&P446L zygotes
are fully viable and fertile.
![]() |
Discussion |
---|
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---|
The significant conformational change due to the exchange of Pro446 to Leu
in the Ketel protein is further supported by the S317T suppressor mutation
that restores Ketel gene function. In human importin-ß the
corresponding Ser311 (in the linker region between HEAT repeats 7 and 8) and
Pro441 are 32.5 Å apart and yet the SerThr exchange in the
Drosophila homologue restores function of importin-ß
(Fig. 9). The 10 Å area
surrounding serine is hydrophobic. The stronger hydrophobicity of threonine
compared with serine does perhaps increase apolar interactions and bend the
molecule back to its functional structure.
The P446L mutation changes Ran binding ability
Experiments with digitonin-permeabilized HeLa cells show that, to a reduced
extent, the P446L proteins do participate in formation of the nuclear import
complexes and in their docking to the cytoplasmic surface of the NE; however,
they do not support import of the complexes into the nuclei in the presence of
Ran, energy source, RanGAP and RanBP1. In fact, the import complexes do not
form upon the addition of the latter components. Apparently the main
structural domains of the P446L protein are intact (binds importin-,
NPC and Ran) but the interaction with Ran is altered. Indeed, we found that
the binding of wild-type and P446L Ketel proteins to Ran are very different:
the P446L cannot bind to RanGTP, to which the wild-type importin-ß binds
strongly, but shows elevated affinity to RanGDP, to which the wild-type
protein shows very little affinity. It is noteworthy that a single amino acid
exchange outside the classical Ran-binding domain can change Ran binding
ability dramatically. The change in Ran-binding ability is most likely the
source of the KetelD-associated dominant female sterility.
However, the KetelD-associated dominant-negative effect is
not manifested via nuclear protein import but rather through the prevention of
cleavage nuclei formation: revealing a novel importin-ß function.
The P446L mutant importin-ß exerts its toxic effect at the end
of mitosis
The injection experiments into wild-type cleavage embryos revealed that the
P446L mutant protein does not inhibit nuclear protein import: when co-injected
with P446L, a fluorescent nuclear substrate readily entered the nuclei.
Furthermore, although the cleavage nuclei enter mitosis and the chromosomes
segregate normally, intact NE never forms in the presence of P446L mutant
importin-ß. Failure of NE assembly in the presence of P446L is revealed
by the following observations. First, the homogenous distribution of (1) a
fluorescent nuclear substrate; (2) the high molecular weight dextrane; and (3)
the GFP-tubulin. Second, the absence of the nuclear lamina lining. Thus the
mutant P446L importin-ß reveals a novel importin-ß function required
during the mitosis-to-interphase transition, a function distinct from the
already known functions of importin-ß in nuclear protein import and in
mitotic spindle assembly (Görlich and
Kutay, 1999; Wiese et al.,
2001
; Nachury et al.,
2001
; Gruss et al.,
2001
).
The P446L mutant importin-ß possesses altered Ran-binding properties:
it does not bind RanGTP but shows elevated affinity to RanGDP. A series of
experiments showed that altered RanGTP-RanGDP balance leads to a similar
phenotype in yeast (i.e. arrest in mitosis-to-interphase transition)
(Sazer and Nurse, 1994;
He et al., 1998
). Results of
enzyme assays described in the present paper show that the altered Ran-binding
ability of P446L importin-ß does not interfere with the GTP hydrolysis
and nucleotide exchange on Ran and thus it is unlikely that the
KetelD-related defects are consequences of distorted Ran
metabolism. Most probably importin-ß is a downstream effector of Ran
during mitosis-to-interphase transition, as in nuclear protein import and
mitotic spindle assembly.
Although several functions of Ran and importin-ß during the cell cycle
were described, the exact molecular mechanisms are still missing. Here we
describe a novel function of Drosophila importin-ß during
mitosis-to-interphase transition where it is involved in the formation of
intact NE. There seem to be three feasible explanations for the
P446L-associated defects. First, since the P446L importin-ß shows higher
affinity to RanGDP than wild-type importin-ß, a possible explanation may
be the depletion of significant amounts of RanGDP that is required for NE
reassembly at the end of mitosis (Zhang
and Clarke, 2000; Hetzer et
al., 2000
). Removal of RanGDP by P446L may lead to the failure of
cleavage nuclei formation. We do not think this explanation is very likely for
the following reasons. (1) Binding and nucleotide exchange assays revealed
that the affinity of the P446L to RanGDP is low and hence depletion of a
significant fraction of Ran from the cytoplasm is rather unlikely. (2)
Interestingly, defects do not evolve in nuclear protein import or in spindle
formation and chromosome segregation following injection of P446L despite the
fact that both nuclear protein import and spindle formation have been shown to
be Ran dependent. Ran's involvement in NE assembly has also been described but
since none of the aforementioned Ran-related processes were disturbed, the
P446L protein does not seem to disturb the Ran cycle. A second possible
explanation of the P446L-related defects is perhaps the inability of the P446L
protein to bind RanGTP and, consequently, the inability to release factors
required for proper chromatin decondensation and/or NE assembly. In this case
the role of importin-ß in the above processes would resemble its function
in mitotic spindle formation, where it is thought to be required for the
release of factors needed for spindle assembly [e.g. NuMA, TPX2
(Wiese et al., 2001
;
Nachury et al., 2001
;
Gruss et al., 2001
)]. A third
possibility is that the P446L-related defects are not associated with the
change in Ran-binding ability. The P446L mutation may disturb the
association of thus far unidentified factors (e.g. nucleoporins). In the case
of the second and third possibilities the factor(s) required for the newly
described importin-ß-related functions remain to be identified.
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Acknowledgments |
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