Cell, Vol. 66, 837-847,
September
6, 1991, Copyright
0 1991 by Cell Press
Cytosolic Proteins That Specifically Bind Nuclear Location Signals Are Receptors for Nuclear Import Stephen A. Adam’+ and Larry Geracet *Department of Cell, Molecular and Structural Biology Northwestern University Medical School Chicago, Illinois 60611 *Department of Molecular Biology Research Institute of Scripps Clinic La Jolla, California 92037
Summary We have purified two major polypeptides of 54 and 56 kd from bovine erythrocytes that specifically bind the nuclear location sequence (NLS) of the SV40 large T antigen. When added to a permeabilized cell system for nuclear import, the purified proteins increase by 2-to C&fold the nuclear accumulation of a fluorescent protein containing the large T antigen NLS. The import stimulation is saturable and dependent upon the presence of cytosol. Nuclear protein accumulation in vitro is sensitive to inactivation by N-ethylmaleimide (NEM). NEM inactivation can be overcome by addition of the purified NLS-binding proteins to the import system. NEM treatment of the purified proteins abolishes their ability to stimulate import but does not affect NLS binding. Our results indicate that the NLS-binding proteins are NEM-sensitive receptors for nuclear import. At least one other NEM-sensitive cytosolic activity and an NEM-insensitive cytosolic activity are also necessary for protein import in vitro. Introduction Communication between the nuclear and cytoplasmic compartments of the cell is mediated by the nuclear pore complex, a large proteinaceous structure of 125 x IO6 daltons that spans the nuclear envelope (Gerace and Burke, 1988; Silver, 1991). The pore complex contains an aqueous channel with a functional diameter of approximately 10 nm, which allows rapid, nonselective diffusion of small molecules between the nucleus and cytoplasm. Passive diffusion through this channel is responsible for the nucleocytoplasmic movement of ions, metabolites, and possibly some small proteins (Paine et al., 1975; see also Breeuwer and Goldfarb, 1990). In contrast, highly selective, mediated mechanisms are involved in transport of most macromolecules across the pore complex, including most nuclear proteins (Feldherr et al., 1984), ribosomal subunits (Bataille et al., 1990) ribonucleoproteins (Stevens and Swift, 1966; Mehlin et al., 1988) and viral nucleoproteins (Dales and Chardonnet, 1973; Tognon et al., 1981; Martin and Helenius, 1991). The mediated import of proteins into the nucleus is specified by short amino acid sequences present in nuclear proteins called nuclear location sequences (NLSs) (Lanford and Butel, 1984; Gerace and Burke, 1988). Most NLSS that have been characterized in detail consist of a stretch
of 3-5 basic residues, often flanked by a proline or glycine. Although no strong consensus has emerged from analysis of this class of NLSs, many contain the sequence Lys-Argl Lys-X-ArglLys (Chelsky et al., 1989). The most extensively studied NLS of this class is that of the SV40 large T antigen, which comprises the sequence Pro-LysizBLys-LysArg-Lys-Val. A single missense mutation of Lys-128 to Thr orAsndramaticallyreducestheefficiencyof thissequence to direct nuclear localization (Lanford and Butel, 1984; Kalderon et al., 1984b), while other mutations in surrounding residues have a lesser effect (Kalderon et al., 1984a). Recently, a more complex NLS has been described for the Xenopus protein nucleoplasmin, consisting of two interdependent basic regions of 2-4 residues each separated by about 10 residues (Robbins et al., 1991). NLSs can function in multiple internal positions within a single protein (Roberts et al., 1987; Nelson and Silver, 1989) although it is possible that the transport efficiency is regulated by the immediate structural context (Rihs and Peters, 1989). An important advance for analyzing the functions of NLSs was the observation that synthetic peptides containing an NLS can direct in vivo nuclear import of large carrier proteins to which they are chemically coupled (Goldfarb et al., 1986; Lanford et al., 1986; Yoneda et al., 1987). This process has characteristics of a receptormediated process, since it is saturable, and since its rate can be decreased by coinjection of free peptide containing a functional NLS but not by peptide containing a mutant nonfunctional NLS. Additional physiological evidence suggests that cytoplasmic receptors interact with NLSs (Breeuwer and Goldfarb, 1990). A number of proteins that interact with NLSs in vitro have been detected by biochemical analysis and are candidates for nuclear import receptors (Adam et al., 1989; Yamasaki et al., 1989; Li and Thomas, 1989; Silver et al., 1989; Lee and Melese, 1989; Imamoto-Sonobe et al., 1990), and several of these components are partially cytoplasmic (Adam et al., 1989; Yamasaki et al., 1989). While NLS-binding proteins of similar molecular weights have been identified in a number of different cells using different approaches, the relatedness of these proteins in unknown. Furthermore, none of these proteins has been shown to be involved in the import process by functional approaches. Interaction of NLS-containing proteins with a cytoplasmic receptor is likely to be an initial step in mediated nuclear import. A subsequent step is suggested to be NLS-dependent binding to the cytoplasmic side of the pore complex, based on in vivo and in vitro studies of nuclear import with colloidal gold probes (Finlay et al., 1987; Newmeyer and Forbes, 1988; Richardson et al., 1988). Afterward, NLS-containing proteins are suggested to interact with a putative “transporter” assembly situated in the central channel of the pore complex (Richardson et al., 1988; Akey and Goldfarb, 1989). Translocation of the bound proteins through the central channel of the pore complex requires ATP and is accompanied by gating of the channel
Cell 838
(Feldherr and Akin, 1990; Newmeyer and Forbes, 1988; Richardson et al., 1988; Akey and Goldfarb, 1989). Channel gating allows protein-coated colloidal gold particles with a diameter of at least 25 nm to be transported across the pore complex (Feldherr et al., 1984; Dworetzky and Feldherr, 1988; Feldherr and Akin, 1990). Ceil-free systems for nuclear protein import promise to be important tools for dissecting the biochemistry of this process. One such system is provided by Xenopus egg extracts, which can assemble nucleus-like structures around naked DNA and reseal the nuclear envelopes of isolated rat liver nuclei. These nuclei selectively accumulate the Xenopus nuclear protein nucleoplasmin and carrier proteins coupled to synthetic peptides containing NLSs (Newmeyer et al., 1988a, 1986b). This system reproduces major features of nuclear import seen in intact cells, including dependence on an intact nuclear envelope and ATP, and inhibition at reduced temperatures and by wheat germ lectin. ATP-dependent association of nuclear proteins with nuclei isolated from mammalian cells (Markland et al., 1987; Imamoto-Sonobe et al., 1988; Parniak and Kennady, 1990) and yeast (Silver et al., 1989; Kalinich and Douglas, 1989) has also been described, although the degree to which these systems reproduce authentic nuclear import is unclear, since the integrity of the nuclear envelope has not been characterized in these cases. While it is clear that components of the nuclear pore complex are involved in protein import (Featherstone et al., 1988; Newmeyer and Forbes, 1988; Davis and Fink, 1990; Nehrbass et al., 1990) there is no agreement on the role of cytoplasmic factors in the process. Newmeyer and Forbes (1990) have shown that cytosolic factors supplied by the Xenopus egg extracts are necessary for nuclear envelope resealing and/or import in that system, while the systems utilizing isolated nuclei apparently have no cytosol requirement (Markland et al., 1987; Imamoto-Sonobe et al., 1988; Silver et al., 1989; Kalinich and Douglas, 1989; Parniak and Kennady, 1990). We recently have described a system involving digitonin-permeabilized cells in order to study the biochemistry of nuclear protein import (Adam et al., 1990). Digitonin treatment of cells perforates the plasma membrane but does not disrupt the nuclear envelope and most other intracellular structures (Adam et al., 1990). Nuclear protein accumulation in this system reflects all the features of mediated import seen in living cells, since it requires functional NLSs, ATP, and an intact nuclear envelope and is inhibited at O°C and by wheat germ agglutinin. This system absolutely requires addition of exogenous cytosol, presumably replacing endogenous cytosolic factors released by digitonin permeabilization. In this study, we have used this permeabilized cell system to study the functions of cytoplasmic NLS-binding proteins that we previously identified. To this end, we have purified these NLS-binding proteins from a cytosol fraction that supports nuclear import. Our results demonstrate that the purified proteins are capable of stimulating nuclear protein accu,mulation in vitro. Furthermore, these proteins can restore the import capacity of cytosol that has been inactivated by pretreatment with N-ethylmaleimide (NEM), and they themselves are inactivated by treatmentwith
NEM. These data indicate that the NLS-binding proteins we have isolated are NEM-sensitive receptors for nuclear import. Results Purification of the NLS Receptor In vitro protein import in digitonin-permeabilized cells requires supplementation of the permeabilized cells with exogenous cytosol (Adam et al., 1990). We originally found that cytosol from a number of different cell types could be used for this assay, including commerically available rabbit reticulocyte lysate. More recently, we observed that erythrocyte lysates function as well as reticulocyte lysates in this import system. We have used erythrocyte lysates for the work described in this study. We decided to purify the NLS-binding proteins from bovine erythrocyte cytosol for functional studies. Our approach was to purify specific NLS-binding proteins, based on their ability to be cross-linked to an NLS peptide. No attempt was made to locate functional activities (i.e., those that stimulate import) during the purification; therefore, additional cytosolic import components, including other putative NLS receptors, would have been missed. Indeed, our results indicate that additional cytosolic components for import are present in the erythrocyte lysates (see below). We detected two NLS-binding proteins of approximately 55 kd in this erythrocyte cytosol, using chemical crosslinking with a radioactively labeled peptide containing the NLS of the SV40 large T antigen (Adam et al., 1989; see Experimental Procedures). These proteins have the same apparent binding specificity as the 56-66 kd NLS-binding proteins (corrected for the mass of cross-linked peptides) we previously detected in cytoplasmic and nuclear fractions of rat liver (Adam et al., 1989; see Figures 1 and 4). The NLS-binding proteins from bovine erythrocytes were purified to homogeneity by conventional chromatography using the peptide cross-linking assay to monitor purification (Figure 1A; see Experimental Procedures). A doublet of about 55 kd were the major bands cross-linked to the NLS peptide in pooled fractions from each of the four major chromatography steps used for the purification (Figure 1A). Several final chromatography steps removed only very minor contaminants and are not shown. The final purified material consisted of two major bands of 54 kd and 56 kd and several minor bands of similar mobility, as seen on a silver-stained gel (Figure 1 B). Cross-linking analysis suggested that all of the protein bands in the cluster specifically bind the radiolabeled peptide (see below). The heterogeneity of the& binding proteins may be due to partial proteolysis durirfg purification, posttranslational modifications of a single protein, or expression of a closely related family of polypeptides with similar function. ApproximateJy 50-100 ug of purified protein typically can be obtained from 1 liter of packed-red cells. Peptide competition experiments were. carried out to demonstrate that these ipolated bovine .erythrocyte proteins bind NLS with the specif&ity expected of an import receptor (Figure 1 C). Utilizing a number of sequences pre-
Receptors 839
for Nuclear Protein Import
C
A 1 2
3
4
MW P
123
45
Buffer
+
Receptor figure
1. Purification
of the NLS Receptor
The receptor was purified by conventional chromatography as described in Experimental Procedures. The autoradiogram in (A) shows pooled fractions from each step of the purification cross-linked to the iodinated NLS peptide Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu Asp: lane 1, (NH&SO, pellet; lane 2, phenyl Sepharose eluate; lane 3, DEAE eluate; Lane 4, first Mono Q eluate. The two major bands migrate closer together in the more purified fractions owing to removal of other abundant proteins that migrate in the same region of the gel. (6) is a silver-stained gel of 0.5 &g of the final purified protein eluted from the second Mono Q column. In (C), the final purified protein was analyzed by cross-linking to the iodinated wild-type peptide. To each sample was added a 50-fold molar excess of an unlabeled peptide corresponding to the following sequences: (lane 1) no additional peptide added; (lane 2) Cys-Pro-Lys-Lys-Lys-Arg-Lys-VaCGlu-Asp (wildtype sequence); (lane 3) Cys-Pro-Lys-Asn-Lys-ArgLys-VaCGlu-Asp (nonfunctional mutant sequence); (lane 4) Cys-Pro-Thr-Lys-Lys-ArgLys-VaCGlu-Asp (functional mutant sequence); (lane 5) Asp-Glu-ValLys-Arg-Lys-Lys-Lys-Pro-Cys (reverse wild-type sequence; nonfunctional for protein import).
viously characterized for their ability to direct import in vivo (Kalderon et al., 1984a; Lanford et al., 1988), we found that only sequences competent to direct a protein to the nucleus were able to compete for binding of the wild-type large T antigen NLS. Mutant large T antigen sequences inactive in import (Lys-128+Asn, lane 3; reverse sequence, lane 5) were unable to compete efficiently for wild-type NLS cross-linking. These results closely resemble our previous studies of the 56-66 kd NLS-binding protein of rat liver, in which we found a correlation between the ability of a sequence to direct import and its ability to compete for binding and cross-linking of the wild-type large T antigen NLS peptide (Adam et al., 1989). We suggest that the bovine proteins are analogous to the rat proteins we previously described. Stimulation of Import by the Purified Receptors We analyzed the ability of the purified NLS-binding proteins to stimulate in vitro nuclear import, using a system consisting of digitonin-permeabilized normal rat kidney (NRK) cells supplemented with exogenous rat erythrocyte cytosol (Figure 2). Import was determined by the nuclear accumulation of a fluorescent protein, allophycocyanin, which had been chemically coupled to synthetic peptides containing the NLS of the SV40 large T antigen (APC-
Figure 2. Stimulation of Nuclear Protein Accumulation Receptor Protein
by the Purified
The in vitro import reaction was carried out with rat erythrocyte cytosol on permeabilized NRK cells. One microgram of the purified protein was added to a 50 ul reaction containing cytosol or import buffer alone containing ATP and an ATP-regenerating system. The mixtures were added to the permeabilized NRK cells for 30 min; the cells were rinsed in import buffer and fixed with formaldehyde prior to observation.
NLS). The assay measures the nuclear accumulation of the fluorescent APC-NLS relative to the initial background fluorescence of the added cytosol containing the APCNLS The nonimported substrate is removed at the end of the incubation, and the amount of accumulation is determined by scanning photographic negatives. From these values, the intranuclear concentrations of APC-NLS were determined from a standard curve of dilutions of APC-NLS (see Experimental Procedures). As observed previously, no binding to cytoplasmic structures is seen in this assay (Adam and Gerace, 1990). The nuclei of the permeabilized NRK cells are capable of accumulating the APC-NLS approximately 30-fold over the input fluorescent protein concentration after 30 min when concentrated cytosol is used in the assay (Adam et al., 1990). However, the cytosol has been diluted in this experiment to reduce total accumulation to g-fold, in order to facilitate detection of possible stimulatory effects by purified NLS-binding proteins (Figure 2, cytosol). When 1 f.tg of the purified NLS-binding proteins was added to the assay mixture, accumulation of the APC-NLS was increased to 22.5fold (Figure 2, cytosol + receptor). However, when the purified proteins were added to the permeabilized cells in import buffer containing ATP and lacking cytosol, no import was detectable (Figure 2, buffer + receptor). The purified NLS-binding proteins therefore are capable of stimulating import, but they can only do so in the presence of cytosol. This suggests that additional components distinct from the NLS-binding proteins are contributed by cytosol. Preincubation of the permeabilized cells with the purified proteins followed by incubation in the presence of
Figure 3. Stimulation
of import by the Purified Receptor Is Saturable
Standard import reactions were carried out with increasing amounts of the purified receptor to cytosol. Import was allowed to proceed for 30 min before the cells were fixed and observed.
cytosol also did not lead to stimulation (data not shown), suggesting that the NLS-binding proteins and cytosolic factors must be present simultaneously for activity. This is a functional demonstration that an NLS-binding protein is involved in nuclear protein accumulation. Based on the specificity of the interaction of the NLS with the purified proteins and the ability of the purified proteins to stimulate nuclear protein accumulation, the binding proteins we have isolated represent receptors for nuclear import. Saturation of Protein Import Stimulation of import by the purified receptor was saturable. This was determined by adding increasing amounts of purified proteins to the assay and determining the amount of APC-NLS accumulated at the end of a 30 min incubation (Figure 3). Even a small amount of the receptors (0.1 pg) was able to stimulate import by approximately 40%, while the system was saturated with the addition of 0.5 trg of protein (corresponding to a final concentration of added protein of 182 nM). Increasing the amount of receptors did not lead to additional increases in the amount of protein imported. Therefore, the amount of receptors already present in the import system appears to be sufficient for import to run at approximately 65% of capacity under the conditions chosen for the assay. Since increasing concentrations of cytosol lead to greater accumulation of fluorescent protein in the nucleus (Figure 2 and data not shown), it seems likely that cytosolic components are rate limiting in this system. The NLS Receptor Is an NEM-Sensitive Component Involved in Import In our initial characterization of the permeabilized cell system for nuclear import, we found that treatment of either cytosol or permeabilized cells with the sulfhydryl alkylating reagent NEM decreased the extent of nuclear protein import to undetectable levels (Adam et al., 1990). We have since found that the degree of import inhibition achieved by treatment of either cytosol or permeabilized cells with NEM is related to the initial “activity” of the cytosol. When highly “active” cytosol is used, low levels of accumulation
can still be detected when either cytosol or permeabilized cells are pretreated with NEM (20%-300/o of the control level of import). We have investigated the ability of the purified NLS receptors to complement the NEM sensitivity of the import system. Under the conditions of these experiments, mock NEM-treated cytosol (see Experimental Procedures) combined with mock NEM-treated cells resulted in a 15.6-fold accumulation of APC-NLS (Table 1, sample A), expressed as 100% import. NEM treatment of the cytosol resulted in a decrease in import to 19% of this control level (Table 1, sample B). However, when saturating amounts of purified receptors were added to NEM-treated cytosol (Table 1, sample C), import was stimulated nearly 3.4-fold to 64% of the control level. The complementing activity of the purified receptors is itself sensitive to NEM, since no stimulation of import was observed when the purified receptors were treated with NEM before addition to an NEM-treated cytosol (Table 1, sample D). Significantly, we have been unable to restore import to control levels by addition of saturating amounts of purified receptors. Similar experiments involved addition of purified NLS receptors to the import system, where permeabilized cells instead of cytosol were treated with NEM. In this case, addition of purified NLS receptors also overcame the NEM inactivation, leading to an approximately 4.7-fold stimulation of import from 28% of control level to 132% (Table 1, samples E and F). As with cytosol, no stimulation was seen when purified receptors were first treated with NEM (data not shown). The observations that purified NLS receptors can complement NEM inactivation of either cytosol or permeabilized cells argue that NLS receptors are present in both fractions. Why must additional receptor be added to restore control levels of import in NEM-inactivated cytosol or cells? Either there are not enough receptors in cytosol or permeabilized cells alone for the system to work efficiently without additional receptors(Figure 3) or NEM-inactivated receptors in cytosol act as an inhibitor of import that can be effectively competed only by addition of large amounts of purified exogenous receptors. Interestingly, the level of import obtained when permeabilized cells alone were treated with NEM was always higher than the level seen when cytosol alone was treated with NEM (Table 1, compare samples B and F). Furthermore, the level of import stimulation by added NLS receptors was always greater in a system containing NEMtreated cells than one containing NEM-treated cytosol. This may reflect a greater contribution of NLS receptors or other NEM-sensitive components to the import system by cytosol than by permeabilized cells. To investigate more directly whether the NLS receptors were the only NEM-sensitive cemponent required for import, we treated both cytosol and permeabilized cells with NEM. This treatment reduced the level of import to less than 5% of control levels (Table 1, sample G) and this inhibition could not be overcome by the addition of mocktreated receptors (Table 1, sample k). A value of 5% or less is equivalent to less tf)an l-fol,d&ccur&fation and is essentially undetectable in the assay. Even the addition of large amounts of untreated receptors was unable to re-
Receptors 841
for Nuclear Protein Import
Table 1. The NLS Receptors
Are NEM-Sensitive
Components
for Protern import
Cytosol
Cells
Receptors
A B C D
Mock NEM NEM NEM
Mock Mock Mock Mock
Mock NEM
100 19 64 17
E F
Mock Mock
NEM NEM
Mock
28 132
G H
NEM NEM
NEM NEM
Mock
September
6, 1991, Copyright
0 1991 by Cell Press
Cytosolic Proteins That Specifically Bind Nuclear Location Signals Are Receptors for Nuclear Import Stephen A. Adam’+ and Larry Geracet *Department of Cell, Molecular and Structural Biology Northwestern University Medical School Chicago, Illinois 60611 *Department of Molecular Biology Research Institute of Scripps Clinic La Jolla, California 92037
Summary We have purified two major polypeptides of 54 and 56 kd from bovine erythrocytes that specifically bind the nuclear location sequence (NLS) of the SV40 large T antigen. When added to a permeabilized cell system for nuclear import, the purified proteins increase by 2-to C&fold the nuclear accumulation of a fluorescent protein containing the large T antigen NLS. The import stimulation is saturable and dependent upon the presence of cytosol. Nuclear protein accumulation in vitro is sensitive to inactivation by N-ethylmaleimide (NEM). NEM inactivation can be overcome by addition of the purified NLS-binding proteins to the import system. NEM treatment of the purified proteins abolishes their ability to stimulate import but does not affect NLS binding. Our results indicate that the NLS-binding proteins are NEM-sensitive receptors for nuclear import. At least one other NEM-sensitive cytosolic activity and an NEM-insensitive cytosolic activity are also necessary for protein import in vitro. Introduction Communication between the nuclear and cytoplasmic compartments of the cell is mediated by the nuclear pore complex, a large proteinaceous structure of 125 x IO6 daltons that spans the nuclear envelope (Gerace and Burke, 1988; Silver, 1991). The pore complex contains an aqueous channel with a functional diameter of approximately 10 nm, which allows rapid, nonselective diffusion of small molecules between the nucleus and cytoplasm. Passive diffusion through this channel is responsible for the nucleocytoplasmic movement of ions, metabolites, and possibly some small proteins (Paine et al., 1975; see also Breeuwer and Goldfarb, 1990). In contrast, highly selective, mediated mechanisms are involved in transport of most macromolecules across the pore complex, including most nuclear proteins (Feldherr et al., 1984), ribosomal subunits (Bataille et al., 1990) ribonucleoproteins (Stevens and Swift, 1966; Mehlin et al., 1988) and viral nucleoproteins (Dales and Chardonnet, 1973; Tognon et al., 1981; Martin and Helenius, 1991). The mediated import of proteins into the nucleus is specified by short amino acid sequences present in nuclear proteins called nuclear location sequences (NLSs) (Lanford and Butel, 1984; Gerace and Burke, 1988). Most NLSS that have been characterized in detail consist of a stretch
of 3-5 basic residues, often flanked by a proline or glycine. Although no strong consensus has emerged from analysis of this class of NLSs, many contain the sequence Lys-Argl Lys-X-ArglLys (Chelsky et al., 1989). The most extensively studied NLS of this class is that of the SV40 large T antigen, which comprises the sequence Pro-LysizBLys-LysArg-Lys-Val. A single missense mutation of Lys-128 to Thr orAsndramaticallyreducestheefficiencyof thissequence to direct nuclear localization (Lanford and Butel, 1984; Kalderon et al., 1984b), while other mutations in surrounding residues have a lesser effect (Kalderon et al., 1984a). Recently, a more complex NLS has been described for the Xenopus protein nucleoplasmin, consisting of two interdependent basic regions of 2-4 residues each separated by about 10 residues (Robbins et al., 1991). NLSs can function in multiple internal positions within a single protein (Roberts et al., 1987; Nelson and Silver, 1989) although it is possible that the transport efficiency is regulated by the immediate structural context (Rihs and Peters, 1989). An important advance for analyzing the functions of NLSs was the observation that synthetic peptides containing an NLS can direct in vivo nuclear import of large carrier proteins to which they are chemically coupled (Goldfarb et al., 1986; Lanford et al., 1986; Yoneda et al., 1987). This process has characteristics of a receptormediated process, since it is saturable, and since its rate can be decreased by coinjection of free peptide containing a functional NLS but not by peptide containing a mutant nonfunctional NLS. Additional physiological evidence suggests that cytoplasmic receptors interact with NLSs (Breeuwer and Goldfarb, 1990). A number of proteins that interact with NLSs in vitro have been detected by biochemical analysis and are candidates for nuclear import receptors (Adam et al., 1989; Yamasaki et al., 1989; Li and Thomas, 1989; Silver et al., 1989; Lee and Melese, 1989; Imamoto-Sonobe et al., 1990), and several of these components are partially cytoplasmic (Adam et al., 1989; Yamasaki et al., 1989). While NLS-binding proteins of similar molecular weights have been identified in a number of different cells using different approaches, the relatedness of these proteins in unknown. Furthermore, none of these proteins has been shown to be involved in the import process by functional approaches. Interaction of NLS-containing proteins with a cytoplasmic receptor is likely to be an initial step in mediated nuclear import. A subsequent step is suggested to be NLS-dependent binding to the cytoplasmic side of the pore complex, based on in vivo and in vitro studies of nuclear import with colloidal gold probes (Finlay et al., 1987; Newmeyer and Forbes, 1988; Richardson et al., 1988). Afterward, NLS-containing proteins are suggested to interact with a putative “transporter” assembly situated in the central channel of the pore complex (Richardson et al., 1988; Akey and Goldfarb, 1989). Translocation of the bound proteins through the central channel of the pore complex requires ATP and is accompanied by gating of the channel
Cell 838
(Feldherr and Akin, 1990; Newmeyer and Forbes, 1988; Richardson et al., 1988; Akey and Goldfarb, 1989). Channel gating allows protein-coated colloidal gold particles with a diameter of at least 25 nm to be transported across the pore complex (Feldherr et al., 1984; Dworetzky and Feldherr, 1988; Feldherr and Akin, 1990). Ceil-free systems for nuclear protein import promise to be important tools for dissecting the biochemistry of this process. One such system is provided by Xenopus egg extracts, which can assemble nucleus-like structures around naked DNA and reseal the nuclear envelopes of isolated rat liver nuclei. These nuclei selectively accumulate the Xenopus nuclear protein nucleoplasmin and carrier proteins coupled to synthetic peptides containing NLSs (Newmeyer et al., 1988a, 1986b). This system reproduces major features of nuclear import seen in intact cells, including dependence on an intact nuclear envelope and ATP, and inhibition at reduced temperatures and by wheat germ lectin. ATP-dependent association of nuclear proteins with nuclei isolated from mammalian cells (Markland et al., 1987; Imamoto-Sonobe et al., 1988; Parniak and Kennady, 1990) and yeast (Silver et al., 1989; Kalinich and Douglas, 1989) has also been described, although the degree to which these systems reproduce authentic nuclear import is unclear, since the integrity of the nuclear envelope has not been characterized in these cases. While it is clear that components of the nuclear pore complex are involved in protein import (Featherstone et al., 1988; Newmeyer and Forbes, 1988; Davis and Fink, 1990; Nehrbass et al., 1990) there is no agreement on the role of cytoplasmic factors in the process. Newmeyer and Forbes (1990) have shown that cytosolic factors supplied by the Xenopus egg extracts are necessary for nuclear envelope resealing and/or import in that system, while the systems utilizing isolated nuclei apparently have no cytosol requirement (Markland et al., 1987; Imamoto-Sonobe et al., 1988; Silver et al., 1989; Kalinich and Douglas, 1989; Parniak and Kennady, 1990). We recently have described a system involving digitonin-permeabilized cells in order to study the biochemistry of nuclear protein import (Adam et al., 1990). Digitonin treatment of cells perforates the plasma membrane but does not disrupt the nuclear envelope and most other intracellular structures (Adam et al., 1990). Nuclear protein accumulation in this system reflects all the features of mediated import seen in living cells, since it requires functional NLSs, ATP, and an intact nuclear envelope and is inhibited at O°C and by wheat germ agglutinin. This system absolutely requires addition of exogenous cytosol, presumably replacing endogenous cytosolic factors released by digitonin permeabilization. In this study, we have used this permeabilized cell system to study the functions of cytoplasmic NLS-binding proteins that we previously identified. To this end, we have purified these NLS-binding proteins from a cytosol fraction that supports nuclear import. Our results demonstrate that the purified proteins are capable of stimulating nuclear protein accu,mulation in vitro. Furthermore, these proteins can restore the import capacity of cytosol that has been inactivated by pretreatment with N-ethylmaleimide (NEM), and they themselves are inactivated by treatmentwith
NEM. These data indicate that the NLS-binding proteins we have isolated are NEM-sensitive receptors for nuclear import. Results Purification of the NLS Receptor In vitro protein import in digitonin-permeabilized cells requires supplementation of the permeabilized cells with exogenous cytosol (Adam et al., 1990). We originally found that cytosol from a number of different cell types could be used for this assay, including commerically available rabbit reticulocyte lysate. More recently, we observed that erythrocyte lysates function as well as reticulocyte lysates in this import system. We have used erythrocyte lysates for the work described in this study. We decided to purify the NLS-binding proteins from bovine erythrocyte cytosol for functional studies. Our approach was to purify specific NLS-binding proteins, based on their ability to be cross-linked to an NLS peptide. No attempt was made to locate functional activities (i.e., those that stimulate import) during the purification; therefore, additional cytosolic import components, including other putative NLS receptors, would have been missed. Indeed, our results indicate that additional cytosolic components for import are present in the erythrocyte lysates (see below). We detected two NLS-binding proteins of approximately 55 kd in this erythrocyte cytosol, using chemical crosslinking with a radioactively labeled peptide containing the NLS of the SV40 large T antigen (Adam et al., 1989; see Experimental Procedures). These proteins have the same apparent binding specificity as the 56-66 kd NLS-binding proteins (corrected for the mass of cross-linked peptides) we previously detected in cytoplasmic and nuclear fractions of rat liver (Adam et al., 1989; see Figures 1 and 4). The NLS-binding proteins from bovine erythrocytes were purified to homogeneity by conventional chromatography using the peptide cross-linking assay to monitor purification (Figure 1A; see Experimental Procedures). A doublet of about 55 kd were the major bands cross-linked to the NLS peptide in pooled fractions from each of the four major chromatography steps used for the purification (Figure 1A). Several final chromatography steps removed only very minor contaminants and are not shown. The final purified material consisted of two major bands of 54 kd and 56 kd and several minor bands of similar mobility, as seen on a silver-stained gel (Figure 1 B). Cross-linking analysis suggested that all of the protein bands in the cluster specifically bind the radiolabeled peptide (see below). The heterogeneity of the& binding proteins may be due to partial proteolysis durirfg purification, posttranslational modifications of a single protein, or expression of a closely related family of polypeptides with similar function. ApproximateJy 50-100 ug of purified protein typically can be obtained from 1 liter of packed-red cells. Peptide competition experiments were. carried out to demonstrate that these ipolated bovine .erythrocyte proteins bind NLS with the specif&ity expected of an import receptor (Figure 1 C). Utilizing a number of sequences pre-
Receptors 839
for Nuclear Protein Import
C
A 1 2
3
4
MW P
123
45
Buffer
+
Receptor figure
1. Purification
of the NLS Receptor
The receptor was purified by conventional chromatography as described in Experimental Procedures. The autoradiogram in (A) shows pooled fractions from each step of the purification cross-linked to the iodinated NLS peptide Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu Asp: lane 1, (NH&SO, pellet; lane 2, phenyl Sepharose eluate; lane 3, DEAE eluate; Lane 4, first Mono Q eluate. The two major bands migrate closer together in the more purified fractions owing to removal of other abundant proteins that migrate in the same region of the gel. (6) is a silver-stained gel of 0.5 &g of the final purified protein eluted from the second Mono Q column. In (C), the final purified protein was analyzed by cross-linking to the iodinated wild-type peptide. To each sample was added a 50-fold molar excess of an unlabeled peptide corresponding to the following sequences: (lane 1) no additional peptide added; (lane 2) Cys-Pro-Lys-Lys-Lys-Arg-Lys-VaCGlu-Asp (wildtype sequence); (lane 3) Cys-Pro-Lys-Asn-Lys-ArgLys-VaCGlu-Asp (nonfunctional mutant sequence); (lane 4) Cys-Pro-Thr-Lys-Lys-ArgLys-VaCGlu-Asp (functional mutant sequence); (lane 5) Asp-Glu-ValLys-Arg-Lys-Lys-Lys-Pro-Cys (reverse wild-type sequence; nonfunctional for protein import).
viously characterized for their ability to direct import in vivo (Kalderon et al., 1984a; Lanford et al., 1988), we found that only sequences competent to direct a protein to the nucleus were able to compete for binding of the wild-type large T antigen NLS. Mutant large T antigen sequences inactive in import (Lys-128+Asn, lane 3; reverse sequence, lane 5) were unable to compete efficiently for wild-type NLS cross-linking. These results closely resemble our previous studies of the 56-66 kd NLS-binding protein of rat liver, in which we found a correlation between the ability of a sequence to direct import and its ability to compete for binding and cross-linking of the wild-type large T antigen NLS peptide (Adam et al., 1989). We suggest that the bovine proteins are analogous to the rat proteins we previously described. Stimulation of Import by the Purified Receptors We analyzed the ability of the purified NLS-binding proteins to stimulate in vitro nuclear import, using a system consisting of digitonin-permeabilized normal rat kidney (NRK) cells supplemented with exogenous rat erythrocyte cytosol (Figure 2). Import was determined by the nuclear accumulation of a fluorescent protein, allophycocyanin, which had been chemically coupled to synthetic peptides containing the NLS of the SV40 large T antigen (APC-
Figure 2. Stimulation of Nuclear Protein Accumulation Receptor Protein
by the Purified
The in vitro import reaction was carried out with rat erythrocyte cytosol on permeabilized NRK cells. One microgram of the purified protein was added to a 50 ul reaction containing cytosol or import buffer alone containing ATP and an ATP-regenerating system. The mixtures were added to the permeabilized NRK cells for 30 min; the cells were rinsed in import buffer and fixed with formaldehyde prior to observation.
NLS). The assay measures the nuclear accumulation of the fluorescent APC-NLS relative to the initial background fluorescence of the added cytosol containing the APCNLS The nonimported substrate is removed at the end of the incubation, and the amount of accumulation is determined by scanning photographic negatives. From these values, the intranuclear concentrations of APC-NLS were determined from a standard curve of dilutions of APC-NLS (see Experimental Procedures). As observed previously, no binding to cytoplasmic structures is seen in this assay (Adam and Gerace, 1990). The nuclei of the permeabilized NRK cells are capable of accumulating the APC-NLS approximately 30-fold over the input fluorescent protein concentration after 30 min when concentrated cytosol is used in the assay (Adam et al., 1990). However, the cytosol has been diluted in this experiment to reduce total accumulation to g-fold, in order to facilitate detection of possible stimulatory effects by purified NLS-binding proteins (Figure 2, cytosol). When 1 f.tg of the purified NLS-binding proteins was added to the assay mixture, accumulation of the APC-NLS was increased to 22.5fold (Figure 2, cytosol + receptor). However, when the purified proteins were added to the permeabilized cells in import buffer containing ATP and lacking cytosol, no import was detectable (Figure 2, buffer + receptor). The purified NLS-binding proteins therefore are capable of stimulating import, but they can only do so in the presence of cytosol. This suggests that additional components distinct from the NLS-binding proteins are contributed by cytosol. Preincubation of the permeabilized cells with the purified proteins followed by incubation in the presence of
Figure 3. Stimulation
of import by the Purified Receptor Is Saturable
Standard import reactions were carried out with increasing amounts of the purified receptor to cytosol. Import was allowed to proceed for 30 min before the cells were fixed and observed.
cytosol also did not lead to stimulation (data not shown), suggesting that the NLS-binding proteins and cytosolic factors must be present simultaneously for activity. This is a functional demonstration that an NLS-binding protein is involved in nuclear protein accumulation. Based on the specificity of the interaction of the NLS with the purified proteins and the ability of the purified proteins to stimulate nuclear protein accumulation, the binding proteins we have isolated represent receptors for nuclear import. Saturation of Protein Import Stimulation of import by the purified receptor was saturable. This was determined by adding increasing amounts of purified proteins to the assay and determining the amount of APC-NLS accumulated at the end of a 30 min incubation (Figure 3). Even a small amount of the receptors (0.1 pg) was able to stimulate import by approximately 40%, while the system was saturated with the addition of 0.5 trg of protein (corresponding to a final concentration of added protein of 182 nM). Increasing the amount of receptors did not lead to additional increases in the amount of protein imported. Therefore, the amount of receptors already present in the import system appears to be sufficient for import to run at approximately 65% of capacity under the conditions chosen for the assay. Since increasing concentrations of cytosol lead to greater accumulation of fluorescent protein in the nucleus (Figure 2 and data not shown), it seems likely that cytosolic components are rate limiting in this system. The NLS Receptor Is an NEM-Sensitive Component Involved in Import In our initial characterization of the permeabilized cell system for nuclear import, we found that treatment of either cytosol or permeabilized cells with the sulfhydryl alkylating reagent NEM decreased the extent of nuclear protein import to undetectable levels (Adam et al., 1990). We have since found that the degree of import inhibition achieved by treatment of either cytosol or permeabilized cells with NEM is related to the initial “activity” of the cytosol. When highly “active” cytosol is used, low levels of accumulation
can still be detected when either cytosol or permeabilized cells are pretreated with NEM (20%-300/o of the control level of import). We have investigated the ability of the purified NLS receptors to complement the NEM sensitivity of the import system. Under the conditions of these experiments, mock NEM-treated cytosol (see Experimental Procedures) combined with mock NEM-treated cells resulted in a 15.6-fold accumulation of APC-NLS (Table 1, sample A), expressed as 100% import. NEM treatment of the cytosol resulted in a decrease in import to 19% of this control level (Table 1, sample B). However, when saturating amounts of purified receptors were added to NEM-treated cytosol (Table 1, sample C), import was stimulated nearly 3.4-fold to 64% of the control level. The complementing activity of the purified receptors is itself sensitive to NEM, since no stimulation of import was observed when the purified receptors were treated with NEM before addition to an NEM-treated cytosol (Table 1, sample D). Significantly, we have been unable to restore import to control levels by addition of saturating amounts of purified receptors. Similar experiments involved addition of purified NLS receptors to the import system, where permeabilized cells instead of cytosol were treated with NEM. In this case, addition of purified NLS receptors also overcame the NEM inactivation, leading to an approximately 4.7-fold stimulation of import from 28% of control level to 132% (Table 1, samples E and F). As with cytosol, no stimulation was seen when purified receptors were first treated with NEM (data not shown). The observations that purified NLS receptors can complement NEM inactivation of either cytosol or permeabilized cells argue that NLS receptors are present in both fractions. Why must additional receptor be added to restore control levels of import in NEM-inactivated cytosol or cells? Either there are not enough receptors in cytosol or permeabilized cells alone for the system to work efficiently without additional receptors(Figure 3) or NEM-inactivated receptors in cytosol act as an inhibitor of import that can be effectively competed only by addition of large amounts of purified exogenous receptors. Interestingly, the level of import obtained when permeabilized cells alone were treated with NEM was always higher than the level seen when cytosol alone was treated with NEM (Table 1, compare samples B and F). Furthermore, the level of import stimulation by added NLS receptors was always greater in a system containing NEMtreated cells than one containing NEM-treated cytosol. This may reflect a greater contribution of NLS receptors or other NEM-sensitive components to the import system by cytosol than by permeabilized cells. To investigate more directly whether the NLS receptors were the only NEM-sensitive cemponent required for import, we treated both cytosol and permeabilized cells with NEM. This treatment reduced the level of import to less than 5% of control levels (Table 1, sample G) and this inhibition could not be overcome by the addition of mocktreated receptors (Table 1, sample k). A value of 5% or less is equivalent to less tf)an l-fol,d&ccur&fation and is essentially undetectable in the assay. Even the addition of large amounts of untreated receptors was unable to re-
Receptors 841
for Nuclear Protein Import
Table 1. The NLS Receptors
Are NEM-Sensitive
Components
for Protern import
Cytosol
Cells
Receptors
A B C D
Mock NEM NEM NEM
Mock Mock Mock Mock
Mock NEM
100 19 64 17
E F
Mock Mock
NEM NEM
Mock
28 132
G H
NEM NEM
NEM NEM
Mock
