EXPERIMENTAL

CELL

RESEARCH

202,355-365

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intracellular Distribution of a Nuclear Localizati RUHONGLI, Department

of Biochemistry,

YANGGU

New York University

SHI,

of Medicine,

School

The transport of proteins into the nucleus requires the recognition of a nuclear localization signal sequence. Several proteins that interact with these sequences have been identified, including one of about 66 kDa. We have prepared antibodies that recognize the 66-kDa nuclear localization signal binding protein (NLSBP) and inhibit nuclear localization in vitro. By immunofluorescence, it is seen that the NLSBP is predominantly cytoplasmic and is distributed peripherally around the nucleus and the microtubule organizing center. There is also a weak pun&ate staining of the surface of the nucleus. Methanol-fixed cells can also be stained directly with fluorescently labeled karyophilic proteins. These stains reveal the same cytoplasmic structures as anti-NLSBP. The expression of the NLSBP is growth dependent. When cells grown to confluence are examined, the cytoplasm& staining is greatly reduced, leaving the punctate nuclear staining as the predominant feature. In serum-starved cells, very little staining of either the cytoplasm or the nucleus can be seen. Upon simulation by the addition of serum, the original cytoplasmic and nuclear envelope staining is restored. Cells grown in the presence of colcbicine or taxol have an altered NLSBP distribution but apparently normal cytoplasmic nuclear transport. 0 1992 Academic Press, Inc.

INTRODUCTION

The transport of most, if not all, nuclear proteins from the cytoplasm into the nucleus requires the presence of a nuclear localization signal (NLS)2 either on the protein itself or on an associated protein [l, 21. NLSs from a number of proteins have been identified, and most contain a stretch of several basic amino acids. For example, the NLS of the SV4Q T-antigen, which was the first NLS identified and has been characterized the 1 To whom reprint requests should be addressed at the above address. Fax: (212) 2636166. * Abbreviations used: ASD, the 2-(p-azidosalicylamido)ethyl-1,3’dithiopropionate radical; NEM, IV-ethylmaleimide; NLS, nuclear localization signal; NLSBP, NLS binding protein; NLS-BSA, BSA conjugated with the NLS peptide; NLS peptide, the peptide CGYGPKKKRKVGG (which includes the SV40 T-antigen NLS).

AND JQHN

0. THOMAS”

550 First Auenue, New York, New York 10016

most extensively, consists of the seven-amino-arid sequence PKKKRKV [3]. The addition of to nonnuclear proteins, either through chemical cross-linking, renders them ~~r~~o~hil~~. The ability of the modified protein to localize ila the nucleus, when either expressed in tra~s~e~t~~ cells, micr jetted, or assayed in an in vitro transport system, i pendent on the number of NLSs that the protein contains [4], and to some extent, the position of the NLS within the protein [5]. In addition to a rn~~~rnal NLS sequence that is necessary ransport, other sequences may have substantial cts on the transport kinetics [6, 71. Several groups have identified with NLS sequences. Yoneda et al. found that antibodies raised against the synt DDDED and EEEDE inhibit the transport of proteins into the nucleus. These antibodies ret teins of 69 and 59 kDa 191. that two proteins from rat interact with the SV4O T-antigen NLS: a pred less abundant one of 70 i et al. [12] identified four rat proteins, r weights of 140 kDa, 100 kDa, 70 kDa, an at interact with a number of different NLSs. From eLa cells, Li an Thomas ]13] detected a single protein of 66 kDa with a pl of about 6. Rio&em NLS binding proteins present in both eytoplasmic and ~~~~e~r fractions. The fraction that is associated with nuclei is weakly bound and does not copurify with nuclear pore complexes. Meier and Blobel [14] have identified 140 and 55 kDa NLS binding proteins from rat cells. ~rn~~~~o~~or~scence studies using antibodies directed against the 449 kDa protein show that it is primarily nucleolar. In yeast, proteins with molecular weights of 70,000~ an [XI, 161 and 67,000 [la, 181 have be identified. Unlike the mammalian proteins which are stributed between the cytoplasm and the neacleus, the yeast proteins pear to be primarily nuclear. The process of nuclear transport can be separated into two stages [19-al]. A nuclear protein ie first recognized by a NLS binding protein, and then, in a second step, it is transported into the nucleus. The second step, but not the first, requires the hydrolysis of ATP, is sen-

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0014-4827192 $5.00 Copyright 0 1992 by Academic Press, Inc. rights of reprodxxzion in any form reserved.

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sitive to decreased temperatures, is blocked by antibodies against nuclear pore proteins, and can be inhibited by wheat germ agglutinin. This binds to nuclear pore glycoproteins and may inhibit nuclear transport by binding to nuclear pores [22]. Featherstone et al. [23] have shown that a monoclonal antibody that recognizes the same nuclear pore proteins that bind wheat germ agglutinin blocks protein import as well as RNA export. A specific nuclear localization factor that is a target of wheat germ agglutinin has not, however, been identified. Akey and Goldfarb [24] have shown that nucleoplasmin binds to at least two regions of the nuclear pore complex, suggesting that more than one binding step may precede the actual translocation of a nuclear protein through the pore. There is a circularly arrayed set of binding sites between 10 and 12.5 nm from the pore center that may be coextensive with wheat germ agglutinin binding sites and a binding site directly over the center of the pore. In addition to these sites that are on the nuclear pore complex, Richardson et al. [19] have shown that thin fibers which bind NLS-containing proteins extend from the nuclear pores into the cytoplasm. The role of these fibers in transporting proteins into the nucleus, however, is not known. Most proteins that are localized in specific organelles or regions of the cell are transported to these regions by specific transport mechanisms. In the case of nuclear proteins, it is not clear how the proteins arrive at the nucleus either from their sites of synthesis or from a general cytoplasmic distribution which many nuclear proteins have during mitosis. In this report we describe fluorescence microscopy studies which show that in exponentially growing human and African green monkey cells some of the NLS binding proteins are on the nuclear surface, but most of them are in cytoplasmic structures that surround the nucleus and extend into the cytoplasm from the microtubule organizing center. The presence of this network suggests that there is a cytoplasmic transport system that functions to actively carry nuclear proteins to the nucleus. The expression of this transport system appears to be regulated; in confluent or serum-starved cells, the cytoplasmic staining is greatly decreased, leaving a punctate staining of the nuclear envelope as the most prominent feature. MATERIALS

AND

METHODS

Materials. Nucleoplasmin was isolated from Xenopus laeuis eggs and conjugated with TRITC or FITC according to Newmeyer et al. [25]. TRITC-BSA conjugates were formed at a ratio of 2 TRITC/ BSA as determined spectrophotometrically. Fluorescent conjugates were cross-linked with either NLS peptide (CGYGPKKKRKVGG, containing the SV40 NLS) or unNLS peptide (CGYGPKNKRKVGG, containing a modified SV40 NLS) which had been iodinated to a low specific activity with lzsI using Iodogen (Pierce Chemical, Rockford, IL) according to the manufacturer’s protocol. The conjugation was accomplished with the cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce Chemical, Rock-

THOMAS ford, IL) following the manufacturer’s instructions. Approximately seven peptides were cross-linked per protein as determined by mobility shift on SDS-PAGE and by the specific activity of samples crosslinked with iz51-labeled peptides. Antibody preparation. Anti-NLSBP antiserum was generated by immunizing rabbits with a 45kDa fragment of the 66-kDa NLSBP. Purification of the NLSBP and fragment was assayed by cross-linking to iz51-ASD-NLS peptide (ASD refers to the 2-(p-azidosalicylamido)ethyl-1,3’-dithiopropionate radical) as described by Li and Thomas [13] followed by SDS-PAGE and autoradiography. HeLa cell nuclei [13] were suspended in 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1.5 mM MgCl,, 0.5 mM DTT, disrupted by sonication, and centrifuged at 100,OOOgfor 30 min in a Beckman TLA100.3 rotor. The supernatant was applied to a monoQ ion-exchange column (LKB, Pharmacia) then washed with 50 mM Tris-HCl, 0.5 mM CaCl,, pH 7.5, followed by a linear 0 to 0.7 M NaCl gradient in the same buffer. Each fraction was assayed as described above, and the fractions containing the 45-kDa fragment, which eluted at about 0.15 M NaCl, colwere pooled. These were applied to a 1 X 40-cm hydroxyapatite umn equilibrated with 10 mM potassium phosphate, pH 6.8. After washing with this buffer, the column was developed with a linear 10 to 500 mM gradient of potassium phosphate, pH 6.8. The 45-kDa protein eluted at a phosphate concentration of about 100 mM. The proteins of the peak fraction (Fig. la, lane 3) were resolved by SDSPAGE, and the 45-kDa band was excised and used for immunization of rabbits. The amount of the fragment obtained varies from preparation to preparation; an optimal yield is 50 Fg from 1 ml of packed HeLa cells. Immunological methods. For immunoblots, cells washed in PBS containing 1 mM PMSF were pelleted, solubilized in SDS-PAGE sample buffer containing 1 mM PMSF, and briefly sonicated to shear released DNA, and the proteins were resolved by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and the blots developed with anti-NLSBP [26]. Immunoadsorption was done with IgG-protein A-agarose complexes formed either with anti-NLSBP serum or with preimmune serum. The proteins were either from a 100,OOOg supernatant of sonically disrupted HeLa cells or from a monoQ ion-exchange column fraction (prepared as described above) that contained both the 66-kDa protein and the 45-kDa fragment. Proteins were labeled with iz5I-ASD-NLS peptide as previously described [13]. In uitro nuclear localization. The effect of antisera on nuclear transport was assessed with an in vitro nuclear transport system based on that of Adam et al. [27]. HeLa cells grown on coverslips were permeabilized with 40 pg/ml digitonin in transport buffer (20 mM Hepes, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT) as described [27]. A transport mixture (35 ~1) containing, in transport buffer, 20% HeLa cytosol (see below), 1 pg/ml TRITC-nucleoplasmin, 1 mM ATP, 5 mM creatine phosphate, and 20 U/ml creatine phosphokinase (Sigma, St. Louis, MO) was applied to the coverslip containing the permeabilized cells. Following a 30-min incubation at 30°C, the coverslip was mounted on a microscope slide in a small amount of transport buffer and immediately observed and photographed with Kodak T-Max 400 film through a Zeiss Axiophot microscope and 63X planapochromat objective. Quantitation of the transport by densitometry of the negatives was as described [27]. HeLa cytosol was prepared as follows. Cells grown in DMEM with 5% calf serum to a density of 5 X lo5 cells/ml were pelleted and washed twice with ice-cold PBS. The cell pellet was resuspended in 1.5 vol of ice-cold deionized water and, after 10 min, homogenized with 10 strokes of a tight-fitting dounce homogenizer. This was centrifuged at 15,000g for 15 min, and the supernatant was centrifuged again at 100,OOOgfor 20 min. The supernatant was then dialyzed overnight against transport buffer and centrifuged again at 100,OOOgfor 20 min to yield a preparation containing about 20 mg/ml protein. This was frozen in small portions and stored in liquid nitrogen.

NUCLEAR

LOCALIZATION

Intracellular localization. To determine the intracellular distribution of NLSBP, African green monkey BSC-1 cells were grown on coverslips, fixed in absolute methanol at -20°C and stained with rabbit anti-NLSBP serum at a dilution of 1:lOO followed by TRITC- or FITC-labeled goat anti-rabbit IgG [28]. Cells double stained with antibody and a karyophilic protein were first fixed and stained with anti-NLSBP as above, then incubated at 37°C for 15 min with 10 pg/mI or the indicated concentration of the desired protein conjugated to peptide and/or TRITC as described above. The stained cells were then washed for 5 min in PBS and observed immediately. Extensive washing at this step leads to the loss of karyophilic protein staining. To examine the growth dependence of NLSBP expression, African green monkey BSC-1 cells grown on coverslips in DME supplemented with 10% bovine calf serum were maintained at confluence for 1 week and then transferred to DME containing 0.25% serum for 70 h to produce serum-starved cells. The cells were stimulated by replating and growing in DME with 10% serum for 20 h. RESULTS

The antibodies used in this study were prepared by immunizing rabbits with a 45-kDa fragment of the 66kDa human NLSBP [13]. The appearance of this fragment was noted during attempts to purify ‘251-NLSBP which had been labeled by label transfer from lz51-ASDNLS peptide [13]. We found it difficult to purify the 66-kDa NLSBP free of several other proteins that have similar molecular weights, and therefore focused on the 45-kDa fragment as a source of pure material for the production of antisera. The amount of the fragment in nuclear extracts is variable and increases with the age of the extract, suggesting that it might be a proteolytic product of the 66-kDa NLSBP. Like the parent 66-kDa NLSBP, the fragment can be cross-linked to a peptide containing the SV40 T-antigen NLS (NLS peptide) but not to unNLS peptide in which Lys,,, is replaced by Asn 1131. This Lys has been shown to be essential for nuclear targeting [29]. Although this cross-linking assay is not suitable for quantitation, it can be used to follow the presence of the protein during purification. Initial assays of crude extracts by cross-linking indicated, in agreement with cross-linking studies of others [ 10, 121, that the NLSBP is present in both nuclear and cytoplasmic fractions. Since the nuclear fraction, from which the NLSBP can be released by sonication, offers a starting preparation of greater purity, it was chosen as the starting point for the purification. As mentioned in a previous report [13], the NLSBP is acidic with a pI of about 6. The 45kDa fragment is apparently rather acidic as well since a relatively high ionic strength is required to elute it from a monoQ anion-exchange column. This column provides the major purification step, yielding the preparation shown in Fig. la, lanes 1 and 2. The protein was further purified by hydroxyapatite chromatography to produce the protein shown in Fig. la, lane 3. The 45kDa protein was separated from the small amount of higher molecular weight contaminants seen in Fig. la, lane 3 by SDS-PAGE, and the excised

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FIG. 1. Production and properties of anti-NLSBP antibodies. (a) Purification of the 45kDa fragment that was used for immunization. Lane 1, (Coomassie blue) and lane 2 (autoradiogram of iz51ASD-NLS peptide conjugate), the peak 45kDa NLSBP-containing fraction eluted from a monoQ ion-exchange column; lane 3, the peak 45kDa NLSBP-containing fraction eiuted from a hydroxylapa,tite column. The immunizing protein was excised from an SDS-PAGE gel. (b) Immunoblots of total African green monkey BSC-1 cells (lane 1) or HeLa cells (lane 2) solubilized in SDS-sample bsuffer containing 1 mM PMSF and briefly sonicated to shear the released DNA. Lane 3 is an immunoblot of a HeLa nuclear supernatant used for the purification of the 45-kDa fragment (Materials and Metbods). (c) Immunoadsorption with anti-NLSBP (lanes P and 2) or preimmune IgG (lane 3) bound to protein A-agarose. Samples in lanes 1 and 3 have been adsorbed from total HeLa cell proteins. The sampIe in lane 2 is from a monoQ ion-exchange fraction that contains both the 66-kDa NLSBP and the 45.kDa fragment. The samples have been labeled by radiolabel transfer from 1251-ASD-NLS peptide [k3;.

band was used to immunize two rabbits. sera with the same titer and specificity when assayed by immunoblots or immunofluorescence. As shown in Fig. lb, when total cellular proteins prepared by lysing either HeLa cells or Af~ic~~ green monkey BSG-1 cells in SDS-P probed on Western blots with 66-kDa protein is recognized. from a rabbit immunized wit protein, no evidence of a freshly lysed cells The presence of a can, however, be seen in ex sence of protease inhibitors, and the fragme lates with time in preparations of sonicated as the one shown in Fig. lb tides with molecular weights between 45,600 and 66,000 are also seen in this lane. These ad~iti~~~l bands are not present in the freshly lysed extracts shown in lanes I and 2, and are therefore most likely partially proteolytic sizes. fragments of intermediate mune sera from the two rabbits produce no ba on immunoblots of dilution as the imHeLa cells when used at the s mune sera, and at high co~cent~~~io~~~ different eLa proteins are recognized by each of the two preimmune sera (not shown). As shown by imm~noadsorptio~ experiments, the 66kDa protein that is recognized by the ~~ti-I~LSBP antibodies interacts with nuclear loca~i~~tio~ signals. The

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NLSBP present in a HeLa cell extract was labeled through a label transfer reaction using 1251-ASD-NLS peptide [13] and then the extract was immunoadsorbed with anti-NLSBP-protein A-agarose. As seen in Fig. lc, a 66-kDa protein is the major 1251-labeled product obtained. If this experiment is performed with a sample that contains both the 66-kDa NLSBP and the 45-kDa fragment (an early fraction from the monoQ column discussed above), both proteins are seen (Fig. lc, lane 2). Neither of these proteins is detected when preimmune serum is used for immunoadsorption (Fig. lc, lane 3). Anti-NLSBP

Inhibits

Nuclear Localization

An in vitro assay based on that of Adam et al. [27] can be used to test the ability of the sera to inhibit nuclear localization, In this assay, the import of exogenously added TRITC-nucleoplasmin into the nuclei of digitonin-permeabilized HeLa cells is measured. The permeabilized cells need to be supplemented by the addition of an energy source in the form of an ATP regenerating system and other factors supplied by incubating the permeabilized cells with a cytoplasmic extract. With this assay, the amount of nucleoplasmin that accumulates in the nucleus is about lo-fold greater in the complete system (Fig. 2a) than in a similar assay in which the cytosolic extract is replaced by transport buffer (Fig. 2b). Within the nucleus, the nucleoplasmin is concentrated in the nucleolar region. The reason for this is not known, but possible explanations are discussed below (Discussion). The quantitative data shown in Fig. 2d are taken from extranucleolar regions of the nuclei. As seen in Figs. 2a-2c, nuclear transport is inhibited by the addition of anti-NLSBP to the cytoplasmic extract that is used to complement the transport system of the permeabilized cells. A titration of the cytoplasmic extract with anti-NLSBP (Fig. 2d) shows that the addition of 0.1~1 of the antiserum to extract prepared from approximately lo4 cells (in 40 ~1) causes a 50% decrease in the amount of TRITC-nucleoplasmin accumulated by the nuclei, and the addition of 0.25 ~1 of serum reduces transport to the level that is observed in the absence of added cytoplasm. When a large amount of serum (4 ~1; data not shown) is added to the cytoplasmic extract, no nuclear accumulation of TRITC-nucleoplasm is detected under these assay conditions. These results can be compared to those obtained with preimmune serum which has little effect, even when this large amount (4 ~1; data not shown) is added. Similar results are obtained with anti-NLSBP and preimmune sera from both of the two immunized rabbits. The complementary experiment, in which the digitonin-permeabilized cells attached to coverslips are treated with anti-NLSBP serum followed by the addition of untreated cytoplasmic extract, has little or no effect on transport (Fig. 2d).

THOMAS

Intracellular

Location of NLSBP

The anti-NLSBP antibodies were used to stain HeLa cells, African green monkey BSC-1 cells, and mouse 3T3 cells by indirect immunofluorescence. Only weak staining is observed with the 3T3 cells, presumably because of low cross-reactivity of the anti-human NLSBP with the mouse protein. HeLa and BSC-1 cells give a similar staining pattern, which is seen most clearly with the BSC-1 cells because of their larger size. As shown in Fig. 3, the antibody reacts primarily with cytoplasmic structures that extend from the nucleus. The distribution is asymmetric and is concentrated in a region near the microtubule organizing center, which can be easily identified in cells that are costained with anti-NLSBP and anti-tubulin antibodies (see below). Although most of the NLSBP in these cells appears to be in the cytoplasm, some punctate nuclear staining can be seen when the focal plane is at the surface of the nucleus (Figs. 3,4, 6, and 7). We have investigated the possibility that this nuclear staining might colocalize with nuclear pores by staining cells with both anti-NLSBP and with monoclonal antibodies that recognize the nuclear pore protein p62 [30], but see little if any correspondence between the staining patterns (not shown). Mitotic cells, which are occasionally seen in the field, are stained uniformly throughout the cytoplasm with no staining of the mitotic chromosomes. The cells shown in this report have been fixed with methanol at -2O’C; fixation at room temperature or with ethanol gives comparable results. Formaldehyde fixation, however, greatly reduces the staining with this antibody. Attempts to stain formaldehyde-fixed cells for immunoelectron microscopy have also been unsuccessful. An identical cytoplasmic staining pattern can be seen when the fixed cells are stained with karyophilic proteins, although the staining of the nuclear surface is not apparent. Staining with TRITC-nucleoplasmin is shown in Fig. 3. These cells have been immunofluorescently stained with anti-NLSBP and FITC-secondary antibodies (Fig. 3a), and then stained directly with TRITC-nucleoplasmin (Fig. 3b). The nucleoplasmin staining colocalizes over the same structures identified with the anti-NLSBP antibody. The same results are obtained when cells are stained with TRITC-secondary antibodies and FITC-nucleoplasmin. Similar results are also seen when cells are doubly stained with antiNLSBP and either TRITC or FITC-labeledStaphylococcus aureus protein A (not shown). Protein A has been previously shown to be karyophilic in HeLa or BSC-1 cells [ 131. These cytoplasmic structures are not stained by several other, nonkaryophilic, proteins including IgG, BSA, and ovalbumin. As an example, Fig. 4 shows cells that have been immunofluorescently stained with anti-NLSBP and FITC-secondary antibodies and then directly stained either with TRITC-NLS-BSA (Figs.

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FIG. 2. Inhibition of nuclear localization by anti-NLSBP antibodies. Nuclear Iocalization is assayed in an in vitro system simiiar to that of Adam et al. [27]. (a) The nuclear localization of TRITC-nucleoplasmin in a complete system using HeLa cells that have been grown on coverslips, permeabilized with digitonin, and reconstituted with a HeLa cell cytoplasmic extract and an ATP regeneration system (see Materials and Methods). (b) The localization when the cytoplasmic extract is replaced by buffer. (c) The inhibition of localization when cytoplasmic extract containing anti-NLSBP (0.1~1 of serum/lo4 cells of extract) is used. The bar represents 20 pm. (d) The effect of adding anti-NLSBP serum (0) or preimmune serum (0) to cytoplasmic extract (extract from 104 cells in 40 ~1) is shown quantitatively. Treating the premeabilized cells with anti-NLSBP (in 40 ~1 of transport buffer) has no effect on nuclear localization when assayed with untreated cytoplasmic extract (m). The data are from densitometry of original negatives of photographs such as (a-c) quantitated as described [27] and are expressed relative to the nuclear accumulation in the absence of antiserum (as shown in (a)).

423-4~) or with TRITC-BSA (Figs. 4d-4f). As with nucleoplasmin, staining with the TRITC-NLS-BSA colocalizes over the same structures identified with the anti-

NLSBP antibody. The TRITC!%A staining, however, gives only a weak and diffuse cytoplasmic staining. Similar results are obtained when cells are stained with

LI, SHI, AND

360

FIG. directly

THOMAS

3, Intracellular distribution of NLSBP. Cells are double stained indirectly with anti-NLSBP and FITC second antibody (a), and with TRITC-nucleoplasmin (b). (c) A phase contrast image. The cells are African green monkey BSC-1 cells; the bar represents 20 pm.

TRITC-ovalbumin or TRITC-nonspecific IgG. When cells are costained with anti-NLSBP and TRITCunNLS-BSA (BSA modified with TRITC and unNLS peptide to the same extent as the TRITC-NLS-BSA), there is some staining coincident with the anti-NLSBP, but the staining is much weaker (Fig. 5). As shown in Fig. 5, the intensity of the TRITC-NLSBSA staining, as determined by the intensity of fluorescence of the most intensely stained region of the image, is dependent on the TRITC-NLS-BSA concentration. The binding is saturable and is half-maximal at about 100 nM NLS-BSA. When this experiment is done with TRITC-unNLS-BSA, about a fivefold higher TRITCunNLS-BSA concentration is required than with TRITC-NLS-BSA (Fig. 5). Cells that have been permeabilized by treatment with digitonin, as is done for the in vitro nuclear localization assay described above, are not stained by anti-NLSBP (not shown). This finding is in line with the observation discussed above that anti-NLSBP treatment of the digitonin-permeabilized cells (as opposed to cytoplasmic extract) has little if any effect on nuclear localization. These results suggest that the NLSBP is weakly associated with the cellular matrix and is removed during the relatively gentle process of digitonin permeabilization. Effect of Colchicine and Tax01 As noted above, the NLSBP is distributed asymmetritally around the nucleus. When cells are double stained with anti-NLSBP and anti-tubulin antibodies, it is seen

that the NLSBP-containing structures extend both from the nucleus and from a region that is near the microtubule organizing center (Figs. 6a and 6b). This region itself, however, gives a relatively weak fluorescence when stained with the anti-NLSBP. The same results are obtained when the cells are immunofluorescently stained with anti-tubulin followed by direct staining with fluorescently labeled karyophilic proteins (not shown). In similar experiments, we have seen no correlation between the distribution of NLSBP and vimentin (not shown). To investigate the possibility that the NLSBP might be associated with microtubules, cells were treated either with colchicine, which disrupts microtubules, or with taxol, which promotes their assembly, and then stained with anti-NLSBP. As shown in Figs. 6c and 6d, when microtubules are disrupted through the action of colchicine, the distribution of the NLSBP is dramatically altered. The NLSBP-associated structures become more diffuse in appearance, and they spread throughout the cytoplasm. In the presence of taxol (Figs. 6e and 6f), the NLSBP-associated structures remain clearly defined, but extend further into the cytoplasm. Also, the asymmetric distribution of the NLSBP that is due to the preferential association of NLSBP with an area around the microtubule organizing center is not present in the taxol-treated cells. These exneriments suggest that NLSBP might be associated, either directly or indirectly, with microtubules. The functional significance of this association, however, is not clear since we find that colchicine does not have an appreciable effect on nuclear localization when

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FIG. 4. Staining with nuclear and cytoplasmic proteins. Cells are double stained, as in Fig. 3, either with anti-NLSBP (a) and TRITCNLS-BSA (b) or with anti-NLSBP (d) and TRITC-BSA (e). The corresponding phase contrast images are shown in (cl and (f). The cells are African green monkey BSC-I cells; the bar represents 20 pm.

assayed in the in vitro system described above. In these experiments, cells growing on coverslips were treated with colchicine for 1 h, then permeabilized and used for nuclear localization assays as shown in Fig. 2, but with buffers containing colchicine. There is no difference in nuclear localization between these cells assayed in the presence of colchicine and normal cells assayed in its absence.

Altered Distribution

and Expression

in

The cells used for the experiments discussed above had been maintained in log phase for several generations. A considerably diffe ization pattern of the NLSBP is seen when cells are stained, with either anti-NLSBP an les, TRITC-nucleoplasmin, or TRITC-NLSen in Figs. 7a and

362

LI, SHI,

NLS-

or unNLS-

TRITC-BSA

AND

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FIG. 5. Dependence of the intensity of NLSBP staining on the concentration of TRITC-NLS-BSA (0) or TRITC-unNLS-BSA (0). The TRITC-NLS-BSA data can be fit to a binding curve (solid line) with a dissociation constant of 0.15 PLM TRITC-NLS-BSA. The data points show the intensity of fluorescence (in arbitrary units) measured over the most intensely stained region of the cell minus the background fluorescence. Each point represents the average of 20 cells.

7b, the cytoplasmic staining is greatly decreased in these confluent cells, leaving primarily a punctate staining on the surface of the nucleus. When confluent cells are maintained in medium containing a low concentration of serum (0.25%), the punctate nuclear staining is also lost; only a background level of staining is seen (Figs. 7c and 7d). When these starved cells are replated in media containing 10% serum, the reticular cytoplasmic staining and the punctate nuclear staining are regained (Figs. 7e and 7f). DISCUSSION We have used two methods to visualize a nuclear localization signal binding protein: immunofluorescence using an antibody directed against a 66-kDa NLSBP and direct staining with fluorescently labeled proteins that contain nuclear localization signals. The authenticity of the antibody as being an anti-NLSBP is supported by the observations that nuclear localization in vitro is inhibited by the antisera, which recognize a protein that can be cross-linked to NLS peptide, and that both staining approaches reveal the same intracellular structures. While the antibodies were prepared by immunizing rabbits with a purified 45kDa fragment of the 66-kDa protein, the 45kDa fragment is either absent or present in very low levels in freshly lysed cells; only the 66-kDa

THOMAS

protein is seen. The 45kDa fragment does accumulate over time, particularly in nuclear extracts. While the fragment most likely results from a limited proteolysis of the 66-kDa protein, the enzymes involved and other details of the mechanism by which this fragment is produced are not clear. In actively growing cells, it appears that a small portion of the NLSBP is distributed over the surface of the nucleus in a punctate pattern. The majority of the protein, however, is in the cytoplasm where it is associated with structures that are concentrated at the periphery of the nucleus and near the microtubule organizing center. Biochemical evidence also suggests that in mammalian cells the NLSBPs are found both in the cytoplasm and in the nucleus [lo, 121. In yeast, however, the NLSBPs are found exclusively in the nuclear fraction of cell extracts [15-181. Antibodies that block the transport of proteins into the nucleus have been prepared by immunizing with either EEEDE or DDDED peptides [8,9]. The antibodies recognize a 69-kDa protein that interacts specifically with nuclear localization signals. Immunofluorescence with these antibodies gives primarily a punctate nuclear staining of rat fibroblast F2408 cells. A punctate cytoplasmic staining that is close to the nucleus is also seen, although it varies from cell to cell. This staining pattern is similar to the pattern that we see when confluent cells are stained with the anti-NLSBP described here (Fig. 6), although different than the staining pattern of rapidly dividing cells. It is not clear whether this is indicative of a different set of NLSBP being recognized by the two antibodies, or differences between the cells used for immunofluorescence. We have not been successful at staining rodent cells with our antibody. Another NLS binding protein, with a molecular weight of 140,000, has been localized to the nucleolus, and it has been suggested [14] that the nucleolus may play a central role in the intranuclear transport of proteins. We also see nucleolar staining of some, but not all, cells with both antiNLSBP and with TRITC-NLS-BSA. Whether this nucleolar staining or the nucleolar concentration of nucleoplasmin that is seen in the in vitro nuclear localization assay (Fig. 2) is related to a functional role of the nucleolus in nuclear transport is not presently clear. The observation that direct staining with fluorescently labeled karyophilic proteins reveals the same structures as does immunofluorescence with antiNLSBP is of particular interest since several NLSBPs with molecular weights between 55,000 and 140,000 have been described [8-171. Since direct staining with NLS-containing proteins does not reveal any more structures than staining with the anti-66-kDa NLSBP, it may be that either the 66-kDa protein is, by far, the major NLSBP of the HeLa and African green monkey BSC-1 cells used in this study, or that the other major

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FIG. 6. anti-NLSBP

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PROTEIN

The effect of colchicine (c, d) and taxol (e, f) on NLSBP distribution. Normal cells are shown in (a, b). Cells zre double stained with (a, c, e) and with monoclonal anti-P-tubulin (b, d, f). The cells are African green monkey BSC-1 cells; the bar represents 20 wm.

NLSBP proteins are associated with the same intracellular structures as the 66-kDa protein. While it is generally accepted that most, if not all, karyophilic proteins enter the nucleus by way of nuclear pore complexes, it is not clear how the proteins get to the nuclear pores. There must be a substantial flow of material to the nucleus given its size and the amount of ribosomal proteins that are transported to the nucleolus for ribosome assembly. It is possible that the 66-kDa NLSBP functions as part of a cytoplasmic transport system that interacts with NLS-containing proteins in the cytoplasm and carries them to the nucleus. One example of such a system, in which a small amount of NLSBP that is weakly or reversibly bound at

or near the nuclear pore is in eq~~~~~b~~~~with NLS associated cytoplasmic structures that contain nu proteins which are in transit, has been suggested [31]. Using an in vitro nuclear transport system, in which liver nuclei are made nuclear transport competent incubating them in an extract from Xepz~pus eggs, Newmeyer and Forbes [31] have strar,ed a requirement for a ~-ethy~ma~eimide )-sensitive soluble factor found in tbe cytoplasm o eggs. This factor appears to function directly probably at an early step since ing of NLS-containing proteins properties of this NEM-sensitive ~~o~e~~ are consistent with it functioning as a corn onent of a sb~tt~i~g nu-

364

LI, SHI, AND

THOMAS

FIG. 7. Growth dependence of NLSBP expression. Cells are confluent (a, b), serum-starved (c, d), or serum-starved and then stimulated by replating at a lower density and growing for 20 h in the presence of 10% serum (e, f). The left panels show cells stained with anti-NLSBP; the right panels are phase contrast images. The cells are African green monkey BSC-1 cells; the bar represents 20 pm.

clear protein carrier. Adam et al. [ll, 271 have also shown that a cytoplasmic NEM-sensitive factor(s) is required for nuclear protein import in digitonin-permeabilized cells. It is possible that these factors may, in some way, be related to the 66-kDa NLSBP or the NLSBP-associated structures that are shown here. There is also a factor that is present in Xenopus egg membrane vesicles which is required for in vitro nuclear transport [25], although it is not clear whether the membrane fraction has a direct effect on nuclear transport, or only plays an indirect role by stabilizing the nuclear envelope or repairing damage caused during the isolation of the nuclei. Since the cytoplasmic structures that are stained with the anti-NLSBP antibody appear to be large enough to sediment in the membrane fraction, it is possible that they could be related to this factor. Nucleoplasmin-coated gold particles injected into oocytes bind to fibers that extend from the nuclear pore

complexes into the cytoplasm [ 191. Since NLS-containing proteins bind to these fibers, it is likely that the fibers are associated with NLSBP, and may be related to either the NLSBP that is seen on the surface of the nucleus or possibly to the NLSBP-associated cytoplasmic structures. The intracellular transport of a number of proteins and organelles involves microtubule-based motors [32]. We have presented some evidence which indicates the NLSBP-containing structures might be associated with regions of the microtubule network, either directly or indirectly. This association however, is not essential for nuclear localization in our in vitro assay, nor is it required for the transport of hsp70 into the nucleus following heat shock [33]. It is interesting that the expression of the NLSBP appears to be regulated. It is present in relatively large amounts in actively growing cells that would be ex-

NUCLEAR

LOCALIZATION

petted to be synthesizing large amounts of nuclear teins and is greatly diminished in quiescent cells. increased expression of NLSBP in rapidly growing might make this protein an attractive target for cancer drugs. We thank Emily Garabedian this work, which was supported Society.

proThe cells anti-

for her interest and assistance with by a grant from the American Cancer

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Received March 30,1992 Revised version received June 29, 1992

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