©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Karyophilic Protein Forms a Stable Complex with Cytoplasmic Components Prior to Nuclear Pore Binding (*)

Naoko Imamoto , Taro Tachibana , Masami Matsubae , Yoshihiro Yoneda (§)

From the (1) Department of Anatomy and Cell Biology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The selective nuclear import of karyophilic proteins is directed by short amino acid sequences termed nuclear location signals (NLSs)()(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.


MATERIALS AND METHODS

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 KHPO, pH 7.2), once in washing buffer (10 m M HEPES, pH 7.3, 110 m M CHCOOK, 2 m M (CHCOO)Mg, 2 m M dithiothreitol) and then lysed in lysis buffer (5 m M HEPES, pH 7.3, 10 m M CHCOOK, 2 m M (CHCOO)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 CHCOOK, 2 m M (CHCOO)Mg, 5 m M CHCOONa, 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 CHCOOK 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 10cpm/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.


RESULTS

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).


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).

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 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.

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.


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 CHCOOK 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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by grants from the Japanese Ministry of Education, Science and Culture and the Nissan Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-6-879-3211; Fax: 81-6-879-3219.

The abbreviations used are: NLS, nuclear location signal; WGA, wheat germ agglutinin; APC, allophycocyanin; bBSA, biotinylated bovine serum albumin; hsc70, 70-kDa heat-shock cognate protein; Ran/TC4, Ras-related nuclear protein.


ACKNOWLEDGEMENTS

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.


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