The importin-ß P446L dominant-negative mutant protein loses RanGTP binding ability and blocks the formation of intact nuclear envelope
Gyula Timinszky1,*,
László Tirián1,*,
Ferenc T. Nagy1,4,
Gábor Tóth2,
András Perczel3,
Zsuzsanna Kiss-László1,
Imre Boros4,
Paul R. Clarke5 and
János Szabad1,
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
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Fig. 1. Effects of the wild-type (A-C) and the P446L mutant importin-ß (D-F)
following their injection, along with a fluorescent nuclear substrate, into
wild-type cleavage embryos. Arrows show the site of injection. Import of the
fluorescent nuclear substrate into the nuclei was followed in a laser-scanning
microscope. The A and D, the B and E and the C and F photographs were taken at
roughly identical stages of the cleavage cycles. The few nuclei shown on F
appeared following diffusion of the fluorescent substrate away from the site
of injection. Bar, 100 µm.
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Fig. 2. Effects of P446L mutant importin-ß on cleavage chromatin following its
injection into wild-type cleavage embryos expressing histone-GFP.
Approximately 200 picolitres P446L protein solution (1.2 µM, approximately
the endogenous importin-ß concentration) was injected into the posterior
end of a wild-type cleavage embryo in which histone-GFP highlighted chromatin.
Chromatin organization was followed in a laser-scanning microscope. Optical
sections from the anterior (A-C) and the posterior (D-F) regions of the same
embryo are shown. While the anterior section was devoid of P446L, the P446L
mutant protein was present at the posterior region. A and D represent
interphase chromatin following P446L protein injection. B and E show
segregating chromosomes. C and F show chromatin during the upcoming
interphase. Note that the nuclei doubled in number and the chromosomes
segregate both at the anterior (control) and posterior (`experimental')
regions of the embryo. Bar, 20 µm.
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Fig. 3. Effect of P446L importin-ß on cleavage mitotic spindle organization.
Wild-type (A-D) or P446L mutant importin-ß (E-H) solution was injected
into cleavage Drosophila embryos expressing tubulin-GFP fusion
protein. While mitotic spindle assembly, elongation and disassembly is not
affected by the injected wild-type (A-C) and P446L (E-G) importin-ß, the
tubulin-GFP protein is homogeneously distributed in the site of P446L
injection indicating the failure of NE assembly (H). Tubulin-GFP is excluded
from the nuclei and appear as dark holes on the optical sections (D). Bar, 10
µm.
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Fig. 4. Localization of the chromatin (as revealed by GFP-tagged histone; A,C) and
the red-fluorescent 170 kDa TRITC-dextrane (B,D) in cleavage embryos injected
with wild-type (A,B) or with P446L mutant importin-ß (C,D). Following the
injection of wild-type importin-ß, the TRITC-dextrane is excluded from
the nuclei that form following mitosis (B). However, following the injection
of P446L, the TRITC-dextrane is not excluded from the region of the chromatin
following mitosis (D), an indication of the absence of functional NE. Note
that the chromatin morphology is hardly affected (C). Bar, 20 µm.
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Fig. 5. Cleavage embryos expressing lamin-GFP were injected at the posterior end
(arrows) with wild-type (A-C) or P446L mutant importin-ß (D-F). A and D
show localization of lamin-GFP during interphase following injection. The
lamin-GFP molecules highlight the NE. Embryos are in metaphase in B and E, and
the spindle envelopes are not affected (B,E). During the upcoming interphase,
nuclear lamina re-forms in the embryos that were injected with normal
importin-ß (C). No nuclear lamina assembles at the site of injection of
the P446L mutant importin-ß revealing the failure of intact NE formation
(F). Bar, 50 µm.
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Fig. 6. Nuclear import complexes form and dock on the NE of the
digitonin-permeabilized HeLa cells following the addition of either wild-type
(A) or P446L mutant importin-ß (C) in the presence of the fluorescent
IBB-nucleoplasmin fusion protein. Upon addition of the import mixture (the
fluorescent IBB-nucleoplasmin fusion protein, Ran, NTF2, RanGAP, RanBP1 and
energy supply) and the wild-type importin-ß, nuclear import complexes
form and enter the nuclei (B). However, when P446L mutant importin-ß is
added along with the import mixture, import complexes do not form and the HeLa
cell nuclei are not highlighted by fluorescent signal (D). Bar, 10 µm.
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Fig. 7. RanGDP removes higher amounts of importin-ß from extracts of
KetelD eggs than from extracts of wild-type (WT) eggs.
RanQ69L protein binds higher amounts of importin-ß protein from WT egg
extract than from extract of KetelD eggs. GST protein was
used as a negative control (A, left). RanQ69L protein removes high amounts of
purified WT importin-ß but not purified P446L mutant protein. At the same
time RanGDP removes higher amounts of purified P446L importin-ß compared
with the purified WT importin-ß (A, right). More Ran is precipitated with
the anti-Ketel antibody from extracts of the KetelD eggs
than from extracts of WT eggs. (B, left). However, if an energy-regenerating
system and 3 µM (10 times the endogenous importin-ß concentration in
the extract) purified wild-type or P446L mutant importin-ß are added to
WT egg extract, more Ran is precipitated from the extract supplemented with WT
importin-ß (B, right). Wild-type importin-ß inhibits both exchange
of the labeled GTP from Ran (C) and GTP hydrolysis (D), whereas P446L mutant
importin-ß has no effect on both nucleotide exchange and GTP hydrolysis.
In C, the time course of nucleotide exchange is shown on a semi-logarithmic
scale and D shows the results of the reactions performed in duplicate.
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Fig. 8. CD spectra of model peptides representing wild-type (A) and P446L mutant
importin-ß (B). The CD spectra were recorded in 100% trifluoro-ethanol
(TFE, continuous lines), in a mixture of 66% TFE and 33% H2O
(dashed lines), and in 33% TFE and 66% H2O (dotted lines).
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Fig. 9. Computer modeling of the structure of model peptides (A,C) and
importin-ß (B,D) with Pro (A,B) and Leu (C,D) in the linker region
between HEAT repeats 10 and 11. In computing the structure of the peptides and
the proteins only three angles were changed as shown on the figure. Leu439 and
Leu440 are Ile444 and Ile445 in Drosophila. Pro, Leu and Ser in the
critical position appear dark blue, light blue and yellow, respectively.
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© The Company of Biologists Ltd 2002
