fraction and localization of a novel protein, ISP36, tocompartments in the interchromatin space Masashi Segawa1, Koko Niino1, Reiko Mineki2, Naoko Kaga2, Kimie Murayama2, Kenji Sugimoto3
Trang 1fraction and localization of a novel protein, ISP36, to
compartments in the interchromatin space
Masashi Segawa1, Koko Niino1, Reiko Mineki2, Naoko Kaga2, Kimie Murayama2, Kenji Sugimoto3, Yuichi Watanabe4, Kazuhiro Furukawa1,5and Tsuneyoshi Horigome1,5,6
1 Natural Science Course, Graduate School of Science and Technology, Niigata University, Japan
2 Division of Proteomics and Biomolecular Science, Biomedical Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan
3 Division of Applied Biochemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Japan
4 Department of Biology, Niigata University, Japan
5 Department of Chemistry, Niigata University, Japan
6 Center for Transdisciplinary Research, Niigata University, Japan
In the interior of the cell nucleus, individual reactions
contributing to nuclear function occur at ‘specific
domains’, rather than in the whole nuclear interior To
generate these specific domains, higher order
organiza-tion of the nuclear interior is necessary Each
chromo-somal DNA is anchored to a specific ‘chromosome
territory’ and does not mix with other chromosomal
DNA in the nucleus [1] It is known that proteins
important for particular nuclear functions are not
spread throughout the whole nuclear interior, but are
rather localized to specific compartments as speckles,
meshwork structures and so on [2] Within the
compartments, proteins participating in a specific func-tion are often present as a complex [2] It is also known that many protein complexes are recruited to, stored in and function in interchromosomal ments [2] Precise control of these subnuclear compart-ment structures is crucial for nuclear functions
The highly dynamic and flexible structures of the nuclear interior compartments are based on the nuc-lear envelope which is composed of nucnuc-lear mem-branes, nuclear pore complexes (NPC) and the nuclear lamina Disruption of the integrity of the nuclear lam-ina by mutation of lamin A⁄ C causes Emery-Dreifuss
Keywords
ISP36, interchromatin space protein, nuclear
matrix protein, nuclear protein proteome,
insoluble protein proteome
Correspondence
T Horigome, Department of Chemistry,
Faculty of Science, Niigata University,
Igarashi-2, Niigata 950-2181, Japan
Fax: +81 25 262 6160
Tel: +81 25 262 6160
E-mail: thori@chem.sc.niigata-u.ac.jp
(Received 27 March 2005, revised 4 July
2005, accepted 5 July 2005)
doi:10.1111/j.1742-4658.2005.04847.x
A rat liver nuclear insoluble protein fraction was analyzed to investigate candidate proteins participating in nuclear architecture formation Proteins were subjected to two-dimensional separation by reversed-phase HPLC in 60% formic acid and SDS⁄ PAGE The method produced good resolution
of insoluble proteins One hundred and thirty-eight proteins were separ-ated, and 28 of these were identified The identified proteins included one novel protein, seven known nuclear proteins and 12 known nuclear matrix proteins The novel 36 kDa protein was further investigated for its sub-nuclear localization The human ortholog of the protein was expressed in Escherichia coliand antibodies were raised against the recombinant protein Exclusive localization of the protein to the nuclear insoluble protein frac-tion was confirmed by cell fracfrac-tionafrac-tion followed by immunoblotting Immunostaining of mouse C3H cells suggested that the 36 kDa protein was a constituent of an insoluble macromolecular complex spread through-out the interchromatin space of the nucleus The protein was designated
‘interchromatin space protein of 36 kDa’, ISP36
Abbreviations
DAPI, 4¢,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; GFP, green fluorescent protein; ISP36, interchromatin space protein of 36 kDa; NPC, nuclear pore complex; PMSF, phenylmethylsulfonyl fluoride; TFA, trifluoroacetic acid.
Trang 2muscular dystrophy and familial partial lipodystrophy
[3,4] Mutations of an inner nuclear membrane protein,
emerin, also cause Emery-Dreifuss muscular dystrophy
[4] These observations suggest that structural
compo-nents of the nuclear framework are important for the
structural integrity and functions of the nucleus [3,4]
By contrast, inner nuclear structures have been
sup-posed to be maintained by nuclear matrix filaments [5],
but such filaments have not yet been clearly
demon-strated in vivo and remain under debate [6,7] Some
nuclear proteins present in the interior, such as actin
[8], lamins [9,10], NuMA [11,12] and the
NPC-associ-ated proteins Nup153 [13] and Tpr [14], are known to
form filaments or dot-like structures in the interior
of the nucleus However, whether these proteins are
responsible for the inner nuclear structure is not yet
clear
Recently, many integral nuclear membrane proteins
have been found by proteome analyses, and some of
these are thought to be linked to a variety of
dystro-phies [15] These findings strongly suggest that the
structure and functions of the nuclear envelope are
more complicated than previously expected from
lim-ited protein members Similar to the nuclear envelope,
it is likely that many unknown proteins participate in
maintaining the inner nuclear structures such as
chro-mosomal territories, interchrochro-mosomal compartments
and many kinds of speckles Therefore, searches for
and analyses of novel nuclear structural proteins are
necessary to gain further insight into the inner nuclear
structure, nuclear compartmentalization and higher
order nuclear structure and functions
In this study, we analyzed a rat liver nuclear
insol-uble protein fraction to search for candidate novel
nuclear structural proteins High resolution over a
wide molecular mass range of insoluble proteins was
achieved by two-dimensional separation using
poly-mer-based reversed-phase HPLC in 60% formic acid
and SDS⁄ PAGE The separated proteins were
identi-fied by partial amino acid sequencing or MS, and
con-sisted of novel proteins along with many known
structural proteins One of these novel proteins was
shown to be located to compartments in the
interchro-matin space in the nucleus The protein is suggested to
be a constituent of an insoluble macromolecular
com-plex spread throughout the interchromatin space
Results
We previously developed a reversed-phase HPLC
sys-tem using polymer-based columns for membrane
pro-tein separation [16] Membrane fractions often contain
some proteins that are strongly bound to the columns
and can not be eluted by any solvents other than a strong alkaline solution Therefore, we optimized the separation conditions for membrane proteins by using
a polymer-based column in the presence of 60% for-mic acid [16] The column can be washed with an alka-line solution We applied this method to the separation
of a rat liver nuclear insoluble protein fraction as fol-lows Rat liver nuclei were separated by sucrose density gradient centrifugation and treated with RNase A⁄ DNase I to remove chromatin The obtained fraction contained the nuclear envelope and some of the nuclear matrix proteins This fraction was further extracted with 2% (v⁄ v) Triton X-100 ⁄ 0.3 m KCl to remove lipids and proteins bound to the nuclear insol-uble proteins by ionic and hydrophobic interactions The obtained ‘nuclear insoluble fraction’ was then dis-solved in 100% formic acid, applied to a Poros 10R1 column and eluted with a linear gradient of n-butanol
in 60% (v⁄ v) formic acid The elution profile is shown
in Fig 1A The collected fractions were further separ-ated by SDS⁄ PAGE (Fig 1B) One hundred and thirty-eight components, including a protein as large as
217 kDa, were separated Selected protein bands derived from 21 mg of the nuclear insoluble fraction were excised from the gel and digested with trypsin Next, the obtained peptides were separated by octyl-silica reversed-phase HPLC and sequenced using a protein sequencer One example is shown in Fig 2A Nineteen samples were analyzed by Edman degrada-tion and 15 samples could be identified from the obtained partial amino acid sequences and the mole-cular masses estimated by SDS⁄ PAGE (Table 1) Pep-tides derived from samples < 10 lg were determined
by peptide mass finger printing (Table 1) Eighteen samples were analyzed using the method and 14 sam-ples could be identified In the case of band 2 in Fig 1B, the partial amino acid sequences were further confirmed by MS⁄ MS analysis (Fig 2B) As shown in Table 1, many filament proteins were identified in the nuclear insoluble protein fraction The major compo-nents found in Fig 1B, i.e bands 7, 8, 13, 17, 19, 23 and 25, were lamin A, lamin C, lamin B1, vimentin, keratin type 2, keratin type 1 and actin, respectively, which are all filament proteins Uricase (band 15 in Fig 1B) probably represented a contaminating protein derived from peroxisome cores, because peroxisome cores are composed of uricase protein crystals, often contaminate heavy subcellular fractions such as rough microsomes and are not solubilized by mild detergents [17] We found a novel protein of 36 kDa, correspond-ing to bands 2 in Fig 1B A cDNA sequence encodcorrespond-ing the 36 kDa protein was found in the Universal Protein Resource database (UniProt accession no Q96QD9)
Trang 3However, the protein had not yet been characterized,
although it was referred to as ‘putative 40-2-3 protein’
Therefore, we further determined the localization of
this protein in the nucleus as described below The
other proteins are discussed in detail in the Discussion
Predicted amino acid sequences for the murine,
human and bovine 36 kDa protein (no 2 in Table 1)
were found in the UniProt database The amino acid
sequence of the human ortholog is shown in Fig 3A
We designated the protein as ‘interchromatin space
protein of 36 kDa’ (ISP36), because the protein was
shown to be localized to compartments in the
inter-chromatin space by the results presented below ISP36
is a basic protein with a calculated isoelectric point of
12.5, and contains nuclear localization signal motifs, a
helix-turn-helix DNA-binding motif and an
intermedi-ate filament protein-like structure as shown in Fig 3A
The amino acid sequences of the human, mouse and
bovine ISP36 were very similar, as shown in Fig 3B
Therefore, we used human ISP36 to raise antibodies
against the protein Human ISP36 was expressed as a
His6-tagged protein in Escherichia coli and purified by
Ni-agarose affinity chromatography in the presence of
8 m urea because the expressed protein formed
inclu-sion bodies (Fig 4A) The purified protein was used to
raise antibodies in rabbits The generated antiserum
reacted with the antigen, i.e His6-tagged human ISP36
(Fig 4, B3) The antiserum was then applied to the rat
liver nuclear insoluble fraction from which ISP36 was
originally purified As shown in Fig 4B, a 36 kDa
protein was clearly stained with the anti-ISP36 serum
These results indicate that the antibodies raised against human ISP36 cross-react with rat ISP36, and that ISP36 is really present in the rat liver nuclear insoluble fraction Next, we determined the subcellular localiza-tion of ISP36 using the antibodies and the rat liver subcellular fractions As shown in Fig 5A, ISP36 was localized to the nuclear fraction (Fig 5A, ‘Anti ISP36’ lane 4) Moreover, the protein was completely localized
to the insoluble fraction among the subnuclear frac-tions (Fig 5B, ‘Anti ISP36’ lane 8)
As shown in Fig 5A, affinity-purified anti-ISP36 specifically reacted with ISP36 Therefore, we applied the antibodies to immunostaining of cultured cells When mouse C3H cells were stained with affinity-puri-fied anti-ISP36, the cell nuclei were specifically stained with speckles (Fig 6) When the ISP36 staining was compared with the DNA staining, the ISP36 speckles were dense in regions where the DNA was weakly stained (Fig 6, 2 and 3) When immunoglobulin frac-tions prepared from the preimmune rabbit were used instead of the affinity-purified anti-ISP36, no staining was observed (Fig 6, 1) These results suggested that ISP36 was present in compartments in the interchro-matin space When green fluorescent protein (GFP)– ISP36 was transiently expressed in mouse C3H cells, the fusion protein was completely localized to the nuc-lei (Fig 7) and appeared as speckles similar to the immunostaining of ISP36 in Fig 6 Within the nuc-leus, the protein again seemed to be localized to compartments in the interchromatin space (Fig 7)
To further confirm the localization of ISP36, an
inter-Fig 1 Elution profile of a nuclear insoluble
fraction from a Poros-HPLC column (A) A
rat liver nuclear insoluble fraction (7 mg)
was applied to a Poros 10R1 column
(7.5 · 75 mm, 10 lm porous polystyrene
beads; PerSeptive Biosystems, Cambridge,
MA, USA) buffered with solvent A (60%
for-mic acid, 0.1% TFA, v ⁄ v) and eluted with a
140-min linear gradient from 5 to 40% of
Solvent B (33% n-butanol, 60% formic acid,
0.1% TFA, v ⁄ v) at 1.5 mLÆmin)1 Next, the
concentration of Solvent B was brought to
100% in 5 min The eluate was fractionated
as indicated (B) 1 ⁄ 100 volumes of the
frac-tions were analyzed by 8.5% SDS ⁄ PAGE,
and the gel was stained with silver The
positions of the molecular mass standards
are shown on the left The numbered
pro-tein bands were identified by amino acid
sequencing or MS after tryptic digestion.
Trang 4chromatin granule cluster protein, SC35, was costained
with ISP36 (Fig 8) The antibody staining patterns
were processed using a deconvolution method to
improve the images Most of the SC35 speckles (green)
were colocalized with the ISP36 staining (red) in the
interphase cell nuclei, such that the color became
yel-low (Fig 8) These results confirmed the presence of
ISP36 in compartments in the interchromatin space
To precisely examine the localization of ISP36 in the
cell cycle, we observed cell nuclei in the different
cell-cycle stages (Fig 9) As shown in Fig 9, chromatin
stained by 4¢,6-diamidino-2-phenylindole (DAPI; blue) and ISP36 stained with anti-ISP36 (red) were com-pletely separate within the nucleus When two daugh-ter cells in early G1 phase were observed from the upper side, chromatin and ISP36 were present as speckles without overlapping (Fig 9, panel 1) These staining patterns showed that ISP36 may be con-strained as a constituent of an insoluble macromole-cular complex spread throughout the interchromatin space In prophase, ISP36 staining was partly lost
in the interchromatin regions but accumulated in a limited area (Fig 9, panel 5) In prometaphase and metaphase, ISP36 staining was excluded from the interchromatin region and found in the periphery of condensed chromosomes (Fig 9, panels 6 and 7) Some of the ISP36 did not diffuse into the cytoplasm and remained near the chromosomes These results suggest that ISP36 may still be a constituent of some macro-structure In anaphase, most ISP36 disappeared from the near-chromosomal region (Fig 9, panel 8) Taking the results of the immunostaining and GFP– ISP36 expression together, we conclude that ISP36 is a protein present in compartments in the interchromatin space and may be a constituent of an intranuclear structure that is spread throughout the sinusoidal interchromatin space
Discussion
We used a perfusion-type polystyrene resin column to separate the nuclear insoluble protein fraction in the presence of 60% (v⁄ v) formic acid because the system exhibits high resolution for membrane protein separ-ation [16] In addition, column maintenance is easier than for a silica-based column, because the column can be cleaned with 0.5 m NaOH and still maintain high resolution The column showed high resolution in the separation of nuclear insoluble proteins (Fig 1) Protein recovery was expected to be high, because recoveries of > 70% have been shown previously, even for membrane proteins > 140 kDa [16] Partial amino acid sequencing with a protein sequencer and a peptide mass finger printing method using MS were applied to identify the proteins separated by this method (Table 1) If greater amounts of starting material are used, more proteins of lower abundance may be identi-fied because the reverse-phase HPLC system can easily
be scaled up This method should therefore be applic-able to other proteome analyses of insoluble protein fractions
The proteins identified in this study could be classi-fied into five groups as shown in Table 1 Group A contains proteins that are known to be nuclear matrix
Fig 2 Identification of proteins by partial amino acid sequencing
(A) and MS (B) (A) Twenty micrograms of protein 7 in Fig 1B was
digested in-gel with trypsin, and the generated peptides were
applied to a reversed-phase HPLC equipped with an octylsilica
col-umn (4.8 · 250 mm; Capcel Pack, Shiseido, Tokyo) The peptides
were eluted with a linear gradient of 5–75% (v ⁄ v) acetonitrile
con-taining 0.1% (v ⁄ v) TFA at 0.4 mLÆmin)1 The amino acid sequences
of the separated peptides numbered 1–3 were determined using
a 470A Protein Sequencer (Applied Biosystems) (B) Three
micro-grams of protein 2 in Fig 1B was digested in-gel with trypsin, and
the tryptic peptides were analyzed using an API QSTAR Pulsar
(Applied Biosystems) equipped with a micro-HPLC The total ion
chromatogram and amino acid sequences estimated by MS ⁄ MS
analysis are shown.
Trang 5proteins Filamentous proteins listed in Table 1, i.e.
lamin B1, lamin A, lamin C, vimentin, keratin types 1
and 2 and actin, were all included in this group
Lamins are known to form a meshwork structure
underneath the inner nuclear membrane and function
to maintain the nuclear architecture [3] The majority
of vimentin may be bound to the nuclear lamina from
the outside the nucleus [18] The presence of actin in
the nucleus has been demonstrated by immunogold
staining with partial digestion of the surface lamina of
the nuclear matrix to allow penetration of the gold
particles into the nuclear matrix [19] and the export
mechanism from the nucleus has been well character-ized [20] Nuclear actin may be involved in the regula-tion of nuclear processes, such as chromatin remodeling [21] LAP1 is an intrinsic inner nuclear membrane protein that participates in anchoring the nuclear lamina to the inner nuclear membrane [22] ZAN 75 protein was recently reported to associate with the nuclear matrix [23] and function as a tran-scriptional activator [24] Group B contains proteins that are known to be present in the nucleus, but have not yet been reported to be present in the nuclear mat-rix fraction It was recently reported that a protein
Table 1 Proteins identified by partial amino acid sequencing or MS The proteins identified in the rat liver nuclear insoluble fraction are sum-marized Each ‘No.’ corresponds to that in Fig 1B ‘UniProt Accession no.’ shows the accession number in the Universal Protein Resource Database ‘Molecular mass’ values were obtained from the amino acid sequences ‘S’ and ‘M’ under ‘Identification’ indicate identification by partial amino acid sequencing with a protein sequencer and peptide mass finger printing, respectively ‘No of peptides’ indicates the number
of peptides of which partial amino acid sequences were determined Proteins 2, 20 and 28 were further confirmed by amino acid sequen-cing via nanoelectrospray ionization and quadrupole time-of-flight mass spectrometry Grouping of identified proteins A, Proteins that are known to be nuclear matrix proteins; B, proteins that are known to be nuclear proteins; C, proteins that have not previously been reported
to be present in the nucleus; D, proteins derived from contaminating organelles, namely peroxisomes; and E, novel proteins found in this study.
No Protein
UniProt Accession no.
Molecular mass (kDa) Identification
No of peptides
Sequence coverage (%) Group
P63259
a Accession no in the NCBI protein database.
Trang 6complex containing Nup160 functions to anchor the NPC to the chromatin during NPC reconstitution in late anaphase to early telophase in mitosis, because depletion of the protein complex from a nuclear recon-stitution system caused loss of NPC in the reconstitu-ted nuclear envelope [25] NPC protein 98 binds to the protein complex [25] Therefore, these proteins seem to have requisite functions for NPC reconstitution on chromatin Another group B protein, heterogeneous nuclear ribonucleoprotein G, functions in alternative splicing [26] Interleukin enhancer-binding factor 3 binds protein-arginine methyltransferase and contains double-stranded RNA-binding motifs [27] It is not yet known whether these insoluble nuclear proteins partici-pate in nuclear architecture formation Further charac-terization of these proteins will prove interesting Group C contains proteins that have not previously been reported to be present in the nucleus ATP-bind-ing cassette, subfamily C, member 2 (mrp2) is a mem-ber of the multidrug resistance protein family [28] Most members of this family of proteins are located only in plasma membrane, but one member is also located in intracellular vesicles [28] Actin-binding pro-tein ACF7, neural isoform 2, is a member of the dystonin subfamily and the beta-spectrin superfamily
Fig 3 Amino acid sequence of human ISP36 (A) The amino acid
sequence of human ISP36 (GenBank Accession no AJ344096).
Motifs found in the sequence by programs, ‘ PROFILE ’ and ‘ MOTIF
FIN-DER ’, were underlined (B) Amino acid sequence identities between
human ISP36 and its orthologs The percentages of identical and
similar amino acids are indicated.
Fig 4 Preparation of an anti-ISP36 serum (A) Expression of human His 6 -ISP36 in E coli and its purification using Ni-agarose beads A1 and A2 are lysates of E coli transformed with the pET28c plasmid encoding His6-ISP36 before and after ISP36 induction, respectively A3 con-tains 1 lg of His 6 -ISP36 purified by Ni-agarose beads A1–A3 are stained with Coomassie Brilliant Blue A4–A6 are the same as A1–A3 except that the samples were blotted onto a nitrocellulose sheet, incubated with anti-His sera and then alkaline phosphatase-conjugated sec-ondary antibodies, and developed with 5-bromo-4-chloro-3-indolyl phosphate (B) Purified His6-ISP36 (B1–B3) and a rat liver nuclear insoluble fraction (B4–B6) were electrophoresed and stained with Coomassie Brilliant Blue (B1 and B4) or transferred to a nitrocellulose sheet (B2, B3, B5 and B6) followed by immunostaining For the immunostaining, nitrocellulose strips were incubated with 200-fold diluted anti-ISP36 serum (B3 and B6) or preimmune serum (B2 and B5) and then alkaline phosphatase-conjugated anti-(rabbit IgG), and developed as in (A) The arrowheads indicate ISP36 The bars at the left of panels indicate the positions of marker proteins of 97, 66, 43 and 29 kDa from top to bottom.
Trang 7[29] This protein functions in cytoplasmic microtubule
dynamics to facilitate actin–microtubule interactions
[29] The majority of this protein is located in
cytoplas-mic filaments [29], and whether a proportion of this
protein is localized to the nucleus to interact with
nuc-lear actin needs to be examined
Group D contains a protein that appears to be a
contaminant from peroxisomes as mentioned in the
Results section [17].Group E in Table 1 contains the
36 kDa protein The36 kDa protein was characterized
in this study and designated ISP36 ISP36 was
locali-zed to compartments in the interchromatin space
(Figs 6–9) The interchromatin space starts at the
nuc-lear pores [30], and expands between chromatin
terri-tories and into their interior [1] The surfaces of
compact chromatin domains provide a functionally
relevant barrier that can be penetrated by single
pro-teins or small protein aggregates, but not by larger
macromolecular complexes above a certain threshold
size [1] Therefore, ISP36 is suggested to be a
constitu-ent of an insoluble macromolecular complex spread throughout the interchromatin space, because: (a) the protein was fractionated into the nuclear insoluble fraction in the native state (Fig 5); (b) the protein was
A
B
Fig 5 Subcellular localization of ISP36 (A) Rat liver cytosol (A1),
microsome (A2), mitochondria (A3) and nuclear (A4) fractions were
electrophoresed, transferred to nitrocellulose sheets and then
incu-bated with preimmune IgG fraction or affinity-purified anti-ISP36
sera After incubation with peroxidase-conjugated secondary
anti-bodies, the sheets were developed with H 2 O 2 and
diaminobenzi-dine (B) A rat liver nuclear fraction was treated with DNase–RNase
and the solubilized fraction (chromatin fraction, B5) was separated
by centrifugation The pellet was extracted with 0.5 M NaCl (salt
extract fraction, B6) and 2% (v ⁄ v) Triton X-100, 0.3 M KCl (Tx ⁄ KCl
extract fraction, B7), followed by separation of the insoluble fraction
(insoluble fraction, B8) These factions were immunoblotted as in
(A) The details of the fractionation are described in the
Experimen-tal Procedures Molecular mass markers are same as in Fig 4.
Fig 6 Immunostaining of mouse C3H cells with anti-ISP36 serum Mouse C3H cells cultured on glass coverslips were treated with preimmune IgG fraction (1) or affinity-purified anti-(human ISP36) sera (2 and 3) and then stained with Cy3-conjugated anti-(rabbit IgG) After counterstaining of DNA with DAPI, the cells were observed by fluorescence microscopy The round structure in the DAPI staining corresponds to a cell nucleus Bar, 10 lm.
Fig 7 Localization of GFP–ISP36 protein transiently expressed in mouse C3H cells A plasmid harboring GFP-tagged ISP36 cDNA was transfected into mouse C3H cells and the GFP–ISP36 protein was transiently expressed The cells were immobilized, and the DNA was stained with DAPI and observed by fluorescence micros-copy The round structure in the DAPI staining corresponds to a cell nucleus Bar, 10 lm.
Trang 8localized to compartments in the interchromatin space
by anti-ISP36 staining (Figs 8 and 9); (c) the
immuno-staining did not expand into the chromosomal
com-partment or the cytoplasm (Figs 8 and 9); and (d)
some ISP36 did not diffuse into the cytoplasm and
remained near chromatin in metaphase (Fig 9, panel
7) The interchromatin space contains other
macro-molecular complexes that are required for replication,
translation, splicing and repair [1,31] Therefore, ISP36
may also participate in these nuclear functions We
examined the in vitro DNA binding ability of ISP36
expressed in E coli because the protein contains a
helix-turn-helix DNA-binding motif (Fig 3) ISP36
bound to a nonspecific double-stranded DNA
frag-ment in 0.14 m KCl, although the binding strength
was moderate, i.e bound DNA was dissociated by
0.2 m KCl (data not shown) To elucidate the function
of this novel protein in compartments in the
interchro-matin space, analyses of its dynamics using GFP–
ISP36 and searches for binding proteins both in vivo
and in vitro are necessary as the next step
Experimental procedures
Buffers Buffers were as follows Buffer A: 10 mm Tris⁄ HCl,
pH 7.4, 0.2 mm MgSO4; 8 m urea buffer: 10 mm Tris, 8 m urea, 0.1 m NaH2PO4, pH 8.0; NaCl⁄ Pi: 1.5 mm NaH2PO4, 8.1 mm Na2HPO4, 145 mm NaCl
Animals Animal care, housing and killing were in accordance with the guidelines of the Ministry of Education, Science, Sports and Culture of Japan for the use of laboratory animals
Cell fractionation Subcellular fractions of rat liver were prepared as described previously [32]
Fig 8 Localization of ISP36 and SC35 in mouse C3H cell nuclei.
Mouse C3H cells in interphase were stained by affinity-purified
anti-ISP36 as in Fig 6 (red) An SR splicing factor, SC35, which is
known to be present in interchromatin granule clusters within the
interchromatin space, was stained with an monoclonal anti-SC35
serum (aSC35; BD Biosciences) followed by FITC-labeled
anti-(mouse IgG) (green), and then observed by fluorescence
micros-copy The digital image data of a Z series were captured using a
CCD camera, processed by deconvolution software ( AUTODEBLUR ;
AUTOQUANT ) and presented in 3D ( VOLOCITY , IMPROVISION ) The round
structure corresponds to a cell nucleus Bar, 10 lm.
Fig 9 Intranuclear localization of ISP36 throughout the cell cycle Mouse C3H cells were stained with affinity-purified anti-(human ISP36) sera (red) and DAPI (blue) as in Fig 6, and then observed by fluorescence microscopy The digital image data were processed
as in Fig 8 1, 2: early G 1 ; 3, 4: G 2 to prophase; 5: prophase; 6: pro-metaphase; 7: pro-metaphase; and 8: anaphase 2 and 4 represent side views of stained nuclei Bars, 10 lm.
Trang 9Preparation of rat liver subnuclear fractions
Rat liver nuclei were isolated from fasting rats as described
previously [33] Next, the isolated nuclei were suspended in
50 mm Tris⁄ HCl buffer, pH 7.4, containing 0.25 m sucrose,
5 mm MgSO4, and proteinase inhibitors i.e 1 mm
phenyl-methylsulfonyl fluoride (PMSF), 2 mm benzamidine,
10 lgÆmL)1 of leupeptin and 5 lgÆmL)1 each of antipain,
chymostatin and pepstatin A (150–300 U nucleiÆmL)1) The
amount of material derived from one A260 unit and 1 mL
of isolated nuclei ( 3 · 106
nuclei) was defined as 1 unit (1 U) The suspension was treated with DNase I and
RN-ase A (final concentration of 125 lgÆmL)1for each enzyme)
at 4C for 2 h and then centrifuged at 800 g for 15 min
The supernatant was designated the ‘chromatin fraction’
The pellet was suspended in buffer A (10 mm Tris⁄ HCl,
pH 7.4, 0.2 mm MgSO4) containing protease inhibitors, and
the concentration was adjusted to 167 UÆmL)1 The
suspen-sion was added to 4 vol of buffer A containing 625 mm
NaCl, protease inhibitors and a 1⁄ 25 vol of
2-mercapto-ethanol After incubation for 30 min, the suspension was
centrifuged at 10 000 g for 15 min The supernatant was
designated the ‘salt extract fraction’ The pellet was washed
with buffer A containing 500 mm NaCl and then suspended
in buffer A containing protease inhibitors, followed by
adjustment of the volume to 500 UÆmL)1 The suspension
was added to an equal volume of 40 mm Mes⁄ KCl, pH 6.0,
4% (v⁄ v) Triton X-100, 0.6 m KCl, 20% (w ⁄ v) sucrose,
4 mm EDTA containing protease inhibitors and incubated
for 30 min The suspension was centrifuged at 20 000 g for
30 min The supernatant and pellet were designated the
‘Tx⁄ KCl extract fraction’ and ‘insoluble fraction’,
respect-ively
Purification and identification of proteins in the
nuclear insoluble fraction
Proteins in the rat liver nuclear insoluble fraction were
sep-arated by reversed-phase HPLC in 60% (v⁄ v) formic acid,
followed by SDS⁄ PAGE as described previously [16] For
the view of protein elution pattern, gels were stained by
sil-ver (Fig 1B) Howesil-ver, for further analysis of interesting
proteins, gels were stained with Coomassie Brilliant Blue
and protein bands were excised from the gel The excised
proteins were identified by mass spectrometry or partial
amino acid sequencing using a protein sequencer Samples
obtained in quantities > 10 lg were identified by the
se-quencer Tryptic digestion of the proteins excised from the
gel was performed as described previously [34] The
mix-tures of tryptic peptides were completely dried by vacuum
centrifugation, and then dissolved in 2% (v⁄ v)
trifluoroace-tic acid (TFA) Next, peptide mapping was performed using
a model TSQ 700 triple-stage electrospray ionization
quad-rupole mass spectrometer (Thermo Electron, San Jose, CA,
USA) with a Gilson HPLC system (Gilson, Villiers-le-Bel,
France) The HPLC conditions were as follows: Magic C18 column (0.2· 50 mm; Michrom BioResource, Auburn, CA) eluted with 0.1% (v⁄ v) formic acid (solvent C) and 0.1% (v⁄ v) formic acid in 90% (v ⁄ v) CH3CN (solvent D), using a program of 5% (v⁄ v) solvent D for 5 min, a gradi-ent of 1.05% per min for 90 min and 100% solvgradi-ent D for
5 min, at a flow rate of 2 lLÆmin)1using an Accurate split-ter (1 : 100 v⁄ v; LC Packing, San Francisco, CA, USA) The MS conditions were as follows: ion spray voltage of 2.2–2.7 kV, electron multiplier voltage of 1000 eV, and mass range of 220–2500 (4 s) The proteins were identi-fied using the prowl search engine (http://www.prowl rockefeller.edu/) and the NCBI database The API QSTAR pulsar hybrid mass spectrometer system, consisting of nanoelectrospray ionization and quadrupole time-of-flight, with a microliquid chromatograph (Magic 2002; Michrom BioResource) was used for MS⁄ MS analysis to confirm the amino acid sequences of the peptides The MS conditions for MS⁄ MS analysis were as follows: ion spray voltage of 3.0–3.8 kV, electron multiplier voltage of 2200 V, nitrogen
10 unit curtain gas, nitrogen 10 unit collision gas and colli-sion energy of 20–55 eV
In cases of partial amino acid sequencing, the peptides generated from the protein were purified by octylsilica reversed-phase HPLC and the partial amino acid sequences were determined using a 470A Protein Sequencer (Applied Biosystems) Proteins were identified from their molecular masses and the obtained partial amino acid sequences using the program fasta (http://www.fasta.genome.ad.jp/)
DNA constructs ISP36 cDNA clones for protein expression were prepared using the full-length human ISP36 cDNA sequence A human ISP36 cDNA clone in pSPORT1 (clone ID: DKFZp761B1514; RZPD Deutsches Ressourcenzentrum fur Genomforschung GmbH, Berlin, Germany) was used as
a template for PCR with the following synthetic oligo-nucleotide primers: N, 5¢-CATGAACCGGTTTGGTAC-3¢
as the 5¢ primer, and C, 5¢-CTATCCCACGGTGACAA AGC-3¢ as the 3¢ primer The sequence between these prim-ers was amplified by PCR using Ex Taq DNA polymerase (Takara, Tokyo, Japan) The PCR product was digested with HindIII and XhoI, and the resulting DNA fragment was inserted into the HindIII⁄ XhoI sites of pET28c for His6-tagged protein expression (Novagen, Madison, WI, USA), or the HindIII⁄ SalI sites of pEC2 for GFP-tagged protein expression (Clontech, Mountain View, CA, USA), resulting in pEGFP-ISP36
Expression and purification of His6-ISP36 The pET28c expression plasmid encoding His6-ISP36 was transformed into E coli DE3 (BL21) cells, and the cells were grown in Luria–Bertani medium Expression of the
Trang 10fusion protein was induced by addition of 0.1 mm isopropyl
thio-b-d-galactoside, followed by incubation for 6 h at
30C The bacterial cells were collected and disrupted by
sonication, resulting in the recovery of ISP36 as inclusion
bodies in the precipitate fraction Next, the inclusion bodies
were dissolved in 10 mm Tris, 8 m urea, 0.1 m NaH2PO4,
pH 8.0 (8 m urea buffer), and the resulting supernatant was
loaded onto a column (1.4· 2 cm) packed with 2 mL of
Ni-NTA beads (Qiagen) The column was washed
exten-sively with 8 m urea buffer containing 10 mm imidazole
and the bound His6-ISP36 was then eluted with 8 m urea
buffer containing 1 m imidazole and stored at)80 C until
use
Preparation of rabbit anti-ISP36 sera
An anti-ISP36 serum was raised in female rabbits by
immunization with gel pieces containing His6-ISP36 and
Freund’s adjuvant [32] The IgG fraction was purified from
the antiserum by ammonium sulfate precipitation
Purification of anti-ISP36 sera
His6-ISP36 purified as above (about 3 mg) was dialyzed
against 0.9% (w⁄ v) NaCl, and then precipitated by addition
of 4 volumes of cold acetone The precipitate was dissolved
in 0.3 m triethanolamine⁄ HCl, pH 8.0, 8 m urea, and
reac-ted with 1 mL Tresyl Sepharose 4B (Toso, Tokyo, Japan)
at room temperature for 2–3 days on a rotator After
incu-bation with 0.1 m monoethanolamine, pH 8.0, 8 m urea at
room temperature for 1 h, the affinity resin bearing His6
-ISP36 was thoroughly washed with 20 mm Tris⁄ HCl,
pH 8.0, 0.1% (w⁄ v) SDS, 0.5 m NaCl Immunoglobulin
fractions obtained from the anti-ISP36 serum were
incuba-ted with the affinity resin at 4C overnight After washing
with 20 mm Tris⁄ HCl, pH 7.0, 150 mm NaCl, 0.1% (v ⁄ v)
Triton X-100, the purified antibodies were eluted with
0.1 m glycine⁄ HCl (pH 2.5) and immediately neutralized
with a Tris buffer
Immunoblotting
Samples separated by SDS⁄ PAGE were electrotransferred
onto a nitrocellulose filter The filter was incubated with
1.5 mm NaH2PO4, 8.1 mm Na2HPO4, 145 mm NaCl
(NaCl⁄ Pi) containing 5% nonfat dry milk for 1 h, and then
with the same buffer containing the anti-ISP36 serum
(1 : 200, v⁄ v) for 3 h, followed by three washes in NaCl ⁄ Pi
containing 0.05% (v⁄ v) Tween-20 for 5 min each The filter
was then incubated with horseradish peroxidase-conjugated
goat anti-(rabbit IgG) (1 : 1000, v⁄ v) in NaCl ⁄ Picontaining
5% (w⁄ v) nonfat dry milk for 1 h, washed five times with
NaCl⁄ Pi containing 0.05% (v⁄ v) Tween-20 for 5 min each,
and detected using H2O2and 3,3¢-diaminobenzidine
Cell culture and immunofluorescence Mouse C3H cells were grown in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% (v⁄ v) fetal bovine serum at 37 C in a 5% (v⁄ v) CO2 atmosphere [34] Aliquots of the cells (1–3· 105) were grown on glass coverslips in 35 mm dishes for 48 h, fixed with 4% paraformaldehyde for
20 min and treated with 0.1% (v⁄ v) Triton X-100 for
5 min The fixed cells were incubated with affinity-purified anti-ISP36 sera or preimmune IgG (final concentration,
4 lgÆmL)1), and then with Cy3-conjugated goat anti-rabbit sera (1 : 1500; Jackson Immunoresearch Laboratories, West Grove, PA, USA) After counterstaining with DAPI (1 lgÆmL)1), the cells were observed under a fluorescence microscope (Eclipse E600; Nikon) equipped with a Plan-Apo ·60 objective (NA 1.40; Nikon) Digital images were obtained using luminavision software (Mac version 1.57; Mitani Corp., Tokyo, Japan) controlling an ORCA-ER cooled CCD camera (Hamamatsu Photonics, Shizuoka, Japan) and a Z-axis motor (Ludl Electronic Products Ltd, Hawthorne, NY, USA) for collecting Z-series optical sec-tions (0.1 lm intervals)
Transient expression of GFP-ISP36 The pEGFP-ISP36 plasmid (16 lg) was mixed with 0.6· 107
cells in 0.5 mL of 30 mm NaCl, 120 mm KCl,
8 mm Na2HPO4, 1.5 mm MgCl2 and electroporated into mouse C3H cells in a cuvette with electrodes at 0.4 cm intervals at a setting of 220 V and a 960 lF capacitor using
a Gene Pulser and Capacitance Extender (Bio-Rad, Hercu-les, CA, USA) After the pulse, the electroporated cells were incubated with an equal volume of serum-free MEM (Nissui Pharmaceutical) for 10 min at room temperature, and then aliquots of the cells were grown in an excess amount of Dulbecco’s modified Eagle’s medium on glass coverslips in 35 mm dishes for subsequent microscopic observation
Acknowledgements
This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan and grants from the Biodesign Research Project
of RIKEN and for Project Research of Niigata Uni-versity
References
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