From the
Targeting of karyophilic proteins to nuclear pores is known to
require several cytoplasmic factors, including the nuclear location
signal-binding protein. Using a digitonin-permeabilized cell-free
transport assay, we have obtained a cytoplasmic fraction containing
factors that specifically bind to karyophilic protein and support the
nuclear binding step of the transport. Components in this fraction form
a stable complex with the karyophile through interaction with nuclear
location signal. Since this complex shows nuclear pore binding activity
prior to nuclear entry in the absence of other cytosolic factors, we
call it nuclear pore-targeting complex. It consists of karyophilic
protein and four proteins of 54, 56, 66, and 90 kDa. In our
reconstitution experiments, a complex with 54 and 90 kDa proteins is
capable of targeting karyophiles to the nuclear pores.
The selective nuclear import of karyophilic proteins is directed
by short amino acid sequences termed nuclear location signals
(NLSs)
Previously we showed that cytoplasmic injection of antibodies
against 70-kDa heat-shock cognate protein (hsc70) inhibited nuclear
import in living cells
(8) . Using a digitonin-permeabilized
cell-free transport assay, we confirmed that hsc70 is a necessary but
not sufficient cytoplasmic factor to support active nuclear import of
SV40 T-antigen NLS-containing karyophile
(7) . Therefore, in
this study, we attempted to identify other cytoplasmic factors required
for nuclear import, initially by fractionating cytosolic extracts in
two distinct manners, and examining their ability to support the
nuclear import of SV40 T-antigen NLS (T-peptide) conjugates (T-APC and
T-BSA) in a digitonin-permeabilized cell-free transport assay.
As
one of two fractionation procedures, total cytosol prepared from
Ehrlich ascites tumor cells was fractionated by Q-Sepharose ion
exchange chromatography as described under ``Materials and
Methods.'' Each fraction separated by Q-Sepharose, as well as
hsc70 alone, supported little or no nuclear import by itself
(Fig. 1 A, b-f). However, combination of the two
fractions eluted with 200 m
M KCl and 550 m
M KCl,
termed Q200 and Q550, respectively, reconstituted the nuclear import of
T-peptide conjugates (Fig. 1 A, g). Addition of hsc70
alone to Q200 or Q550, or other combinations of two separated
fractions, did not reconstitute the import (data not shown).
With incubation on ice in the absence
of ATP, the transport substrate accumulated at the nuclear rim in the
presence of unfractionated cytosol. Q550, alone or supplemented with
H1-depleted cytosol, yielded the same rim staining (Fig. 1 C,
a, c, and d). No such rim staining was observed in the
presence of H1-depleted cytosol alone, other fractions eluted from
Q-Sepharose, or transport buffer. In addition, mutant T-peptide
conjugates, with or without Q550, failed to yield the same rim
staining. Thus, it appears that rim staining obtained with Q550 shows
the ATP-independent and NLS-dependent nuclear binding step of the
transport. These results suggest that fraction Q550 contains factors
that bind to karyophilic protein and support the nuclear pore binding
step of transport.
As shown in Fig. 2 A, upon
incubation, T-BSA formed a complex in Q550 that was detectable by
sucrose gradient analysis. No other Q-Sepharose fractions caused a
shift in the T-BSA, indicating that complex formation occurred
specifically in Q550. The complex formation in Q550 was markedly
reduced in the presence of free T-peptide, but to a lesser extent by
mutant T-peptide. We conclude that complex formation was competitively
inhibited by active NLS, although 5
To purify and identify components of the
complex, T-biotinylated BSA (T-bBSA) trapped in immobilized avidin was
incubated with Q550. Four proteins of molecular masses 54, 56, 66, and
90 kDa were eluted with high salt after extensive washing with
transport buffer. All four proteins dissociated specifically from
T-bBSA in the presence of excess free T-peptide (data not shown).
Immunoblotting analysis showed that eluted materials did not contain
both hsc70 and Ran/TC4, although Q550 contained these two molecules
(Fig. 3 A). To determine functionally whether these four
proteins actually constitute the nuclear pore-targeting complex, T-bBSA
was mixed with eluted materials and the mixture was dialyzed and
examined for targeting activity. As shown in Fig. 3, B and C, T-bBSA reconstituted a complex with the four
proteins and showed nuclear binding activity in the absence of other
factors.
At the beginning of this study, we found that Q550
complemented the histone H1-depleted cytosol. Therefore, the four
proteins which reconstituted the protein complex with T-peptide
conjugates were expected to be depleted by histone H1-Sepharose.
Materials eluted with high salt from H1-Sepharose were found to
reconstitute the targeting activity with T-bBSA as shown in
Fig. 4b. Finally, we confirmed that four proteins of
molecular masses 54, 56, 66, and 90 kDa in the eluted materials were
bound to T-bBSA after reconstitution of the targeting activity
(Fig. 4 c, lane 7). These results show that histone
H1-Sepharose depleted all components of the targeting complex from
total cytosol.
In this study, we isolated a nuclear pore-targeting complex
from a fraction designated Q550. Q550 was NEM-sensitive and supported
only the binding step of transport. The addition of histone H1-depleted
cytosol or Q200 was required for complete transport activity. Such
properties of fraction Q550 resemble those of fraction A separated from
Xenopus oocyte extracts by Moore and Blobel
(12) . To
date, NEM-sensitive 54/56-kDa NLS receptor purified from bovine
erythrocytes
(13) , hsc70
(7, 8, 14) ,
and Ran/TC4
(9, 15, 16) have been functionally
identified as factors involved in nuclear protein import. Very
recently, Adam and Adam
(17) reported the identification and
purification of bovine cytosolic NEM-sensitive 97 kDa protein required
for the binding step of transport and that both 54/56-kDa NLS receptor
and 97-kDa protein are necessary for the binding step. Judging from NEM
sensitivity and molecular masses, 54- and 90-kDa proteins identified in
this study might be mouse homologues of bovine 54/56-kDa and 97-kDa
proteins. On the other hand, although Q550 contained hsc70 and Ran/TC4
proteins, these were not components of the nuclear pore-targeting
complex (Fig. 3 A). The 90-kDa protein identified in this
study did not cross-react with rabbit antibodies against the 90-kDa
heat-shock protein (data not shown). Careful experiments must be
carried out to determine whether hsc70 and Ran/TC4 play a role in the
pore-binding step of transport. That is, transient association of hsc70
with NLSs
(8, 18) or targeting complex components may
facilitate the formation of the targeting complex in the cytoplasm or
enhance the pore-targeting efficiency of the complex. Alternatively,
these proteins may be required for an other step in the transport
process.
Histone H1-Sepharose specifically depleted cytosolic
factors that constitute the complex with T-peptide conjugates for the
nuclear-pore targeting. These results support the idea that both
histone H1 and T-peptide conjugates use common transport machinery to
accumulate into the nucleus, although the NLS of histone H1 is unknown.
Since many proteins were bound to histone H1-Sepharose
(Fig. 4 c), it is yet unclear whether the four proteins
identified in this study form a targeting complex with histone H1
directly or with other karyophilic protein(s) associated with histone
H1. It remains unknown whether other kinds of karyophilic proteins such
as karyophiles containing bipartite type NLS and snRNPs form a nuclear
pore-targeting complex with the same components as those of the complex
identified in this study.
WGA is well known to inhibit the
translocation step of transport, but not the initial binding step.
However, in the present study, nuclear rim binding of a transport
substrate was strongly inhibited by WGA in the presence of H1-depleted
cytosol (Fig. 2 C, f) and only slightly inhibited in the
absence of H1-depleted cytosol (Fig. 2 C, c). The inhibitory
effect of WGA on the nuclear pore binding step in vitro was
also reported previously by Adam and Adam
(17) . We suppose that
H1-depleted cytosol contains some factor(s) which dissociates transport
substrate bound at nuclear pores when the subsequent translocation step
is inhibited. The slight inhibition of rim binding by WGA, observed in
the absence of H1-depleted cytosol, may be due to a residual trace of
dissociating activity in the permeabilized cells.
Four proteins of
molecular masses 54, 56, 66, and 90 kDa were found in the
pore-targeting complex formed in Q550. We found that when Q550 was
fractionated by gel filtration before adding transport substrate, no
independent fractions supported pore targeting of T-peptide conjugates
(data not shown). These results indicate that at least two factors are
required for the targeting activity, indicating that both the 54- and
90-kDa proteins identified in this study are essential for
reconstituting the targeting activity. Under our assay conditions,
stimulation of the targeting activity of the complex was not seen when
the complex was formed by adding 56/66-kDa proteins to the 54/90-kDa
proteins. The roles of 56- and 66-kDa proteins in nuclear transport,
which are tightly associated with T-peptide conjugates in an
NLS-dependent manner, remains unclear. Since we used karyophiles
containing multiple NLS peptides per molecule, it is possible that
these two proteins may associate with NLS independently from 54- and
90-kDa proteins and act as different NLS receptors in conjunction with
other cytoplasmic factors present in a fraction other than Q550.
Alternatively, 56- and 66-kDa proteins may act with 54- and 90-kDa
proteins to facilitate complex formation or stabilize the complex once
formed. Further experiments are needed to determine whether these two
proteins actually have a role in nuclear import.
Further
characterization of each component of the targeting complex identified
in this study, together with analysis of its formation, nuclear pore
targeting, and dissociation process, should lead to understanding of
the precise molecular mechanism of nuclear protein import.
We are grateful to Drs. A. Miyata and I. Yahara for
the gift of anti-hsp90 rabbit antibodies and to Drs. H. Seino and T.
Nishimoto for anti-Ran/TC4 antibodies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(1) . The process of mediated
nuclear import is known to involve at least two steps: ATP-independent
binding to the cytoplasmic face of nuclear pores, followed by
translocation through the nuclear pore complex dependent on ATP
hydrolysis
(2, 3) . Wheat germ agglutinin (WGA), which
binds to a family of nuclear pore complex proteins, is known to inhibit
the latter step of transport
(4, 5) . Physiological
evidence has indicated that karyophilic proteins are complexed in the
cytoplasm upon active nuclear import
(6) ; however, no such
complex has yet been identified on a molecular basis. Here we show
in vitro evidence that a karyophilic protein actually forms a
complex with cytoplasmic factors for nuclear pore targeting in the
active nuclear transport process. In view of these results, we propose
that the first step of transport can be divided into two steps:
formation of the pore-targeting complex in the cytoplasm and subsequent
binding of the complex to the nuclear pores.
Cells and Reagents
PtK2 cells were grown and
plated for in vitro assay, as described previously
(7) . Synthetic peptides containing SV40 large T antigen
wild-type NLS (CYGGPKKKRKVEDP: T-peptide) or transport incompetent
point-mutated NLS (CYGGPKTKRKVEDP: mutant T-peptide) were purchased
from Peptide Institute (Osaka, Japan), and WGA from EY
Laboratories, Inc. Histone H1 purified from calf thymus
(8) and
lysozyme (Sigma) were conjugated to CNBr-activated Sepharose 4B
(Pharmacia) by a standard method at a protein concentration of 2 mg/ml
gel.
Preparation of Transport Substrates
For
preparation of biotinylated bovine serum albumin (bBSA), 100 µl of
2 mg/ml sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC-biotin)
(Pierce) dissolved in distilled water was mixed with 800 µl of 2
mg/ml bovine serum albumin (BSA) (Sigma) dissolved in 0.1
M NaHCO, pH 8.5, incubated for 1 h at room temperature,
and dialyzed against 0.1
M potassium phosphate buffer, pH 7.2.
Allophycocyanin (APC) (Calbiochem) and bBSA were conjugated with
T-peptide or mutant T-peptide to produce T-APC, T-bBSA, and mutant
T-APC as described previously
(10) . All the conjugates
contained 10-15 peptides per carrier molecule (analyzed by
SDS-PAGE).
Preparation and Fractionation of Ehrlich Ascites Tumor
Cells Cytosol
Ehrlich ascites tumor cells were freshly harvested
from the abdominal cavity of mice, washed twice in phosphate-buffered
saline (137 m
M NaCl, 2.7 m
M KCl, 8.1 m
M NaHPO
, 1.5 m
M KH
PO
, pH 7.2), once in washing buffer (10
m
M HEPES, pH 7.3, 110 m
M CH
COOK, 2 m
M (CH
COO)
Mg, 2 m
M dithiothreitol)
and then lysed in lysis buffer (5 m
M HEPES, pH 7.3, 10 m
M CH
COOK, 2 m
M (CH
COO)
Mg, 2 m
M dithiothreitol,
20 µ
M cytochalasin B, 1 m
M phenylmethylsulfonyl
fluoride, 1 µg/ml each aprotinin, leupeptin, pepstatin). Extracts
were clarified by sequential centrifugation (1,500
g for 15 min; 15,000
g for 20 min; 100,000
g for 30 min) as described by Adam et al. (11) For total lysate preparation, the clarified extract was
concentrated to 20-40 mg/ml protein concentration by vacuum
dialysis against transport buffer (20 m
M HEPES, pH 7.3, 110
m
M CH
COOK, 2 m
M (CH
COO)
Mg, 5 m
M CH
COONa, 0.5 m
M EGTA, 2 m
M dithiothreitol, 1 µg/ml each aprotinin, leupeptin, pepstatin)
in a collodion apparatus (Schleicher & Schull). For cytosol
fractionation, the clarified extract was applied to Q-Sepharose fast
flow columns (Pharmacia LKB) equilibrated with lysis buffer and eluted
sequentially with CH
COOK free transport buffer containing
200 m
M, 350 m
M, and 550 m
M KCl. Flow-through
fraction and eluted fractions (termed QFT, Q200, Q350, and Q550,
respectively) were dialyzed against transport buffer and concentrated
in the collodion apparatus to uniform protein concentration (10 mg/ml).
For preparation of histone H1-depleted cytosol and control cytosol, 2
volumes of total cytosol was mixed with 1 volume of histone H1 or
lysozyme-conjugated Sepharose and incubated for 2 h at 4 °C.
Unreacted supernatants after centrifugation (7,000 rpm, 1 min) were
used for transport assay.
Sucrose Gradient Analysis
200 µl of Q550
incubated for 2 h on ice with 200 ng of I-labeled T-BSA
(specific activity, 1
10
cpm/µg protein) was
layered over a 7-15% linear sucrose gradient based on transport
buffer. After centrifugation (200,000
g, 12 h, 4
°C in Beckman SW 41Ti), 0.3-ml fractions were collected from the
tube bottoms and respective radioactivities were measured using a
Beckman
counter (model 8000). To examine the specificity of
complex formation, the same experiments were performed with other
Q-Sepharose fractions.
Gel Filtration
200 µl of Q550 incubated with
10 µg of transport substrate (2 h on ice) or reconstituted complex
was applied to Superdex TM200 HR 10/30 connected to a fast protein
liquid chromatography system (Pharmacia LKB). Samples separated in
transport buffer at 0.5 ml/min flow rate were collected into 0.5-ml
fractions and examined for targeting activity. A protein complex formed
in Q550 with either T-APC or T-bBSA was recovered reproducibly in
fraction 24/25.
Reconstitution of the Nuclear Pore-targeting
Complex
To obtain targeting complex components, Q550 was applied
to immobilized avidin (Pierce) precharged with T-bBSA (250 µg
T-bBSA/1 ml gel). After extensive washing with transport buffer,
proteins associated with T-bBSA were either eluted with
CHCOOK free transport buffer containing 2
M NaCl
or with 0-1.5
M NaCl gradient based on transport buffer
containing 25 m
M n-octyl-
-
D-glucoside
(Dojin, Kumamoto, Japan). For reconstitution of the targeting complex,
2 µg of T-bBSA and 100 µg of carrier BSA were mixed with 0.1-ml
aliquots of eluted materials and dialyzed against transport buffer. For
the experiment shown in Fig. 3 C, 30 µg of T-bBSA was
mixed with 0.2 ml of 2
M NaCl-eluted materials containing
30-µg proteins, without adding carrier BSA, and dialyzed against
transport buffer prior to gel filtration. Notably, eluted materials
precipitated when eluted samples were dialyzed against transport buffer
without adding NLS-containing substrates.
Figure 3:
Identification of components of nuclear
pore-targeting complex. A: a, components of the
targeting complex were purified as described under ``Materials and
Methods.'' Silver-stained profile of proteins separated by
SDS-PAGE ( a) and Western blots of the corresponding proteins
incubated either with anti-hsc70 ( b) or anti-Ran/TC4
( c) rabbit serum. Lanes 1, Q550 (10-µg proteins);
lanes 2, materials eluted with 2.0
M NaCl from
immobilized avidin precharged with T-bBSA after incubation with Q550
(2-µg proteins). Arrows indicate positions of hsc70 and
Ran/TC4. B, nuclear binding activity of a complex formed with
materials in A ( a, lane 2) and T-bBSA ( a).
b shows a control experiment in which reconstitution was
carried out without adding the eluted materials (see ``Materials
and Methods''). Photographs were taken and printed with the same
exposure time. C, targeting complex was reconstituted with
materials in A ( a), lane 2, and T-bBSA as
described under ``Materials and Methods'' and isolated by
Superdex TM200 HR 10/30. Each Superdex fraction was examined for
nuclear binding activity as in Fig. 2 B. Proteins in peaks
a and b were analyzed by SDS-PAGE followed by silver
staining. T-bBSA forming a complex with the four proteins (recovered in
peak a) possessed targeting activity, whereas free T-bBSA
(recovered in peak b) showed no targeting activity. The small
portion of the four proteins recovered in peak b, which were
not associated with T-bBSA in this reconstitution experiment, did not
support nuclear binding. All photographs were taken and printed with
the same exposure time. Molecular mass markers were phosphorylase
b, 94 kDa; BSA, 68 kDa; ovalbumin, 43 kDa; and carbonic
anhydrase, 30 kDa. Arrowheads indicate positions of the 90-,
66-, 56-, and 54-kDa proteins.
In Vitro Assays
Transport assays were performed
essentially as described previously
(7, 11) . 10 µl
of testing cytosols containing 100 ng of transport substrates or
isolated, and reconstituted complexes were incubated with permeabilized
cells for 30 min, with or without 1 m
M ATP, 5 m
M creatine phosphate, and 20 units/ml creatine phosphokinase at 30
°C, or on ice, as described in the respective figure legends. After
incubation, cells were fixed with 3.7% formaldehyde in transport
buffer. To visualize T-bBSA localization, the fixed cells were
permeabilized with 0.5% Triton X-100 in phosphate-buffered saline and
incubated with 2 µg/ml fluorescein isothiocyanate-avidin (Pierce)
in phosphate-buffered saline containing 10 mg/ml BSA for 1 h at room
temperature. The samples were then examined using an Axiophot
microscope (Carl Zeiss, Inc.). Staining of nuclear binding was faint
and could be observed clearly only with an oil immersion lens. For
quantitative analysis, the image was focused on a plane corresponding
to a section through the center of the nucleus. Most of the nuclei in
each field of view were usually in the same focal plane. Fluorescence
intensity was quantified by scanning photographic negatives with a dual
wavelength TLC scanner (Shimadzu CS-930).
Other Methods
hsc70 was purified from the
cytoplasm of Ehrlich ascites tumor cells as described previously
(8) . For immunoblotting, proteins were separated by 10%
SDS-PAGE and transferred electrophoretically on to nitrocellulose
sheets. Western blots were probed with anti-hsc70 rabbit serum
(8) or anti-Ran/TC4 rabbit serum
(9) in TBS buffer (20
m
M Tris-HCl, pH 7.5, 0.3
M NaCl) containing 1% skim
milk for 2 h at room temperature after blocking with TBS buffer
containing 3% skim milk for 4 h. Rabbit antibodies were detected with
alkaline phosphatase-conjugated goat antibodies to rabbit IgG (Bio-Rad)
by the standard method.
Figure 1:
Q550 contains
factors that bind to karyophilic protein and support the nuclear
binding step of transport. A, PtK2 cells permeabilized with
digitonin were incubated for 30 min with 10 µl of testing cytosols
containing 1 µg of T-APC at 30 °C in the presence of ATP and
its regenerating system. Testing cytosols were: 10 µl of transport
buffer ( a), 5 µl of transport buffer plus 5 µl of QFT
( b), Q200 ( c), Q350 ( d), Q550 ( e),
and 1 mg/ml hsc70 ( f); 5 µl of Q200 plus 5 µl of Q550
( g) (see ``Materials and Methods'' for fraction
designations). B, Ptk2 cells permeabilized with digitonin were
incubated with 10 µl of testing cytosols containing 1 µg/ml
T-APC as in A. Testing cytosols were: 2 µl of transport
buffer plus 8 µl of total cytosol ( a), histone H1-depleted
cytosol ( b), and control cytosol incubated with
lysozyme-conjugated Sepharose ( c); 8 µl of histone
H1-depleted cytosol plus 2 µl of 1 mg/ml hsc70 ( d), QFT
( e), Q200 ( f), Q350 ( g), and Q550
( h). C, Ptk2 cells permeabilized with digitonin were
incubated for 30 min with 10 µl of testing cytosols containing 1
µg T-APC ( a-d, f) or mutant T-APC
( e) on ice in the absence of energy source. Testing cytosols
were: 2 µl of transport buffer plus 8 µl of total cytosol
( a), histone H1-depleted cytosol ( b), 8 µl of
histone H1-depleted cytosol plus 2 µl of Q550 ( c), 10
µl of Q550 ( d, e) and transport buffer ( f).
Localization of transport substrate was examined by Axiophot microscopy
with a dry lens for the experiments in A and B or
with an oil immersion lens for binding analysis in
C.
The
other procedure was based on previous evidence which indicated that a
small basic karyophilic protein, histone H1, is complexed in the
cytoplasm, and therefore accumulates in the nucleus via mediated import
pathway as do SV40 T-antigen NLS-containing karyophiles, whereas a
small basic non-karyophilic protein, lysozyme, enters the nucleus by
passive diffusion when injected into the cytoplasm of living cells
(6) . Our previous evidence indicated that hsc70 is involved in
nuclear import of both histone H1 and T-peptide conjugates
(8) .
Here, we found that incubation of total cytosol with histone
H1-conjugated Sepharose resulted in depletion of transport activity for
T-peptide conjugates, whereas incubation with lysozyme-conjugated
Sepharose did not (Fig. 1 B, a-c). Among hsc70 alone and
the Q-Sepharose fractions, the addition of fraction Q550 to the
H1-depleted cytosol restored the transport of T-peptide conjugates
(Fig. 1 B, h). Pretreatment of permeabilized cells with
WGA or treatment of Q550 with N-ethylmaleimide (NEM) inhibited
the transport of T-peptide conjugates in all these reconstitution
experiments (data not shown).
10
- to 1
10
-fold excess of free T-peptide over T-peptide in
iodinated T-BSA was required for the inhibition. The requirement for
high concentration of free T-peptide for competition may be due to the
multivalent interaction of the T-peptide conjugate with NLS-binding
protein(s) and/or absorption of free T-peptide by free NLS-binding
proteins in Q550, which may be present in excess over iodinated T-BSA.
Figure 2:
Formation of a nuclear pore-targeting
complex. A, I-T-BSA incubated with transport
buffer containing 10 mg/ml BSA (
) or Q550 (
) was subjected
to sucrose gradient analysis as described under ``Materials and
Methods.'' 10-50% of total
I-T-BSA incubated
with Q550 sedimented toward the tube bottom (sedimented percent varied
between lots of Q550 used).
I-T-BSA incubated with other
Q-Sepharose fractions showed no shift (
). The sum of the
radioactivities sedimented after incubating
I-T-BSA with
Q550 in the presence of 1 or 3 m
M T-peptide ( T) or
mutant T-peptide ( m) was then plotted, with the amount
sedimented in the absence of peptides taken as 100% ( inset
panel). B, T-APC incubated with Q550 was subjected to gel
filtration chromatography using Superdex TM200 HR 10/30 column as
described under ``Materials and Methods.'' Each Superdex
fraction alone was incubated with permeabilized cells on ice for 30
min. Cells were examined with an oil immersion lens as in Fig.
1 C. All photographs were taken and printed with the same
exposure time. Arrowheads indicate elution positions of marker
proteins: a, thyroglobulin, 699 kDa; b, apoferritin,
443 kDa; c,
-amylase, 200 kDa; and d, free
T-APC. Under these conditions, only small amounts of T-APC shifted to
fraction 24/25, whereas more than 90% remained as free T-APC.
C, permeabilized cells were incubated with 8 µl of
fraction 24/25 obtained above and 2 µl of transport buffer ( a,
b, c) or histone H1-depleted cytosol ( d, e, f) in the
presence ( a, c, d, f) or absence ( b, e) of ATP and
its regenerating system at 30 °C for 30 min; transport assays were
performed with permeabilized cells preincubated (on ice, 5 min) with
0.2 mg/ml WGA in transport buffer ( c,
f).
To elucidate the biological significance of this complex, we
isolated the complex by Superdex TM200 HR 10/30 and examined its
nuclear binding activity. As shown in Fig. 2 B, a protein
complex of about 500 kDa containing a transport substrate in fraction
24/25 showed nuclear rim binding activity in the absence of other
cytoplasmic fractions. To confirm whether such rim staining actually
shows the nuclear binding step of transport prior to nuclear entry, we
examined transport in the presence of ATP at 30 °C. As shown in
Fig. 2 C, the transport substrate in the complex in fraction
24/25 showed only nuclear rim accumulation in the presence of ATP. In
contrast, the addition of histone H1-depleted cytosol to fraction 24/25
supported nuclear entry of the transport substrate (assessed by
homogeneous nuclear stainings) only in the presence of ATP. Neither rim
nor homogeneous nuclear staining obtained in the presence of the
H1-depleted fraction was observed upon pretreatment of permeabilized
cells with WGA, whereas rim staining obtained in the absence of
H1-depleted cytosol was only slightly inhibited (compare
Fig. 2C, a, c, and f; discussed below). The use
of fraction Q200 instead of histone H1-depleted cytosol gave the same
results (data not shown). These results showed that in Q550, a
T-peptide conjugate forms a stable complex which binds to the nuclear
pore with no other cytoplasmic fractions and enters the nucleus
dependent on factor(s) present in both H1-depleted cytosol and Q200 and
on ATP hydrolysis. Therefore we term this complex nuclear
pore-targeting complex.
Figure 4:
Depletion of the targeting complex
components from total cytosol by histone H1-conjugated Sepharose. c shows silver-stained profile of proteins separated by SDS-PAGE.
Histone H1-conjugated Sepharose was incubated with total cytosol
( lane 1) as described under ``Materials and
Methods.'' After incubation, the Sepharose was washed with
transport buffer, and materials not bound to histone H1-Sepharose were
collected ( lane 2). The bound materials were then eluted
sequentially with CHCOOK free transport buffer containing
0.2
M NaCl ( lane 3) and 2.0
M NaCl ( lane
4). 1 ml of 0.2
M or 2.0
M NaCl-eluted fraction,
each containing 2-mg proteins, was mixed with 75 µg of T-bBSA and
dialyzed against transport buffer containing 2 µg/ml cytochalasin B
and then the samples were examined for targeting activity. The results
indicated that materials eluted with 2.0
M NaCl ( b),
but not 0.2
M NaCl ( a), from histone H1-conjugated
Sepharose reconstituted the targeting activity with T-bBSA. After the
reconstitution of targeting activity, molecules bound to T-bBSA were
examined by incubating the sample with immobilized avidin (2 h 4
°C). Materials not bound to the immobilized avidin were collected
( lane 5), and the bound materials were then eluted
sequentially with CH
COOK free transport buffer containing
0.2
M ( lane 6) and 2.0
M NaCl ( lane
7). Final materials in lane 7, which were tightly bound
to T-bBSA, contained four proteins of molecular masses 54, 56, 66, and
90 kDa (indicated by arrowheads). Molecular mass markers were
the same as in Fig. 3. Photographs were taken and printed with the same
exposure time.
To determine whether the targeting activity of the
complex depends on the presence of all four proteins, the proteins were
separated by salt gradient elution from T-bBSA trapped in immobilized
avidin. Proteins with molecular masses of 54 and 90 kDa were eluted
slightly faster than the 56- and 66-kDa proteins. Examination of the
targeting activity of protein complexes reconstituted with fractions
containing predominantly either 54/90-, 54/56/66/90-, or 56/66-kDa
proteins revealed that the targeting activity depended on the presence
of 54- and 90-kDa proteins (Fig. 5), indicating that these
proteins can reconstitute the targeting activity.
Figure 5:
Targeting activity depends on the presence
of 54- and 90-kDa proteins. Silver-stained profile of materials eluted
with 0-1.5
M NaCl gradient elution from T-bBSA trapped
by immobilized avidin ( lanes a-f; see ``Materials
and Methods''), and nuclear binding activities of complexes
reconstituted with corresponding materials were examined as in Fig.
3 B. Fluorescence intensity was quantified by scanning
negatives of photographs taken at the same exposure time, as described
under ``Materials and Methods.'' Arrowheads indicate
the position of the 54-, 56-, 66-, and 90-kDa
proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.