MOLECULAR AND CELLULAR BIOLOGY, May 1992, P. 2186-2192 0270-7306/92/052186-07$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 12, No. 5

The Transport of Proteins into the Nucleus Requires the 70-Kilodalton Heat Shock Protein or Its Cytosolic Cognate YANGGU SHI AND JOHN

0.

THOMAS*

Department of Biochemistry, New York University School of Medicine, New York New York 10016 Received 8 November 1991/Accepted 7 February 1992

Most, if not all, karyophilic proteins enter the nucleus through an energy-requiring process that involves the recognition of specific nuclear localization signals. These signals have been identified in a substantial number of nuclear proteins and generally consist of short stretches of basic amino acids (14, 43). For example, the sequence Pro-LysLys-Lys-Arg-Lys-Val is necessary for targeting the simian virus 40 T antigen to the nucleus, and adding it, either chemically or by recombinant methods, to other nonnuclear proteins renders them karyophilic. A clear consensus sequence for nuclear localization signals, however, has not emerged. The transport of proteins to the nucleus occurs through a process that can be experimentally divided into at least two distinct stages (3, 5, 29, 35). In the first stage, nuclear proteins are recognized and brought to nuclear pore complexes, and in a second stage, which requires ATP, the nuclear proteins are translocated through the nuclear pore into the nucleus. The nuclear pore complexes are large structures of about 124 MDa (3, 34) that span both the inner and outer nuclear membranes. They contain a central pore that is large enough to allow the diffusion of proteins that are less than about 40 kDa, but nonetheless, even small karyophilic proteins appear to enter the nucleus facilitated by the receptor-mediated pathway rather than solely by diffusion (5). Proteins larger than 40 kDa can pass through the nuclear pores provided that they contain a nuclear localization signal or are associated with another nuclear localization signalcontaining protein. Even very large signal-containing complexes, such as small nuclear ribonucleoproteins (25) and colloidal gold coated with karyophilic proteins, are transported (19, 35). Several specific nuclear pore complex proteins have been shown to be required for nuclear localization (12). These include members of a group of glycoproteins, the nucleoporins, that contain 0-linked N-acetylglucosamine residues (12). Exactly how these proteins function, however, is not known. The recognition of karyophilic proteins probably involves the interaction between a nuclear localization signal with a nuclear localization signal-binding protein, and several pro*

teins that specifically interact with nuclear localization signals have been identified (14, 43). Some of these proteins are cytosolic, and others are located in the nucleus and nucleolus. It is also possible that some of these proteins shuttle between these organelles as carriers of nuclear proteins. In only one case, however, has an actual involvement in the import process been demonstrated for these proteins (1), and so their exact roles in nuclear transport remain speculative. An involvement of other cytosolic factors is evident. In vitro studies have shown a requirement for at least two proteins that are sensitive to inactivation by N-ethylmaleimide (1, 2, 30). In yeast cells, genetic studies have shown that the NPL1 gene product, a component of the endoplasmic reticulum, is required both for nuclear localization and for the transport of proteins into the endoplasmic reticulum (38). In this report, we present evidence that in HeLa cells, two abundant cytosolic proteins, the 70-kDa heat shock protein hsp70 and its cytosolic cognate hsc70 play an essential role in the transport of proteins into the nucleus. Proteins that are homologous to hsp70 have been found in all species so far examined, from bacteria to mammals (21, 32, 33, 37, 41). There are two major members of the hsp70 family in the cytosol of human cells: a constitutively expressed protein, hsc70, and hsp70, whose synthesis is greatly increased not only as a result of heat shock or other cellular stresses but also during early S phase (26, 27). In addition to these two cytosolic proteins, there are highly related proteins in mitochondria (40) and in the lumen of the endoplasmic reticulum (33). Members of the hsp70 family contain weak ATPase activities that are stimulated by the binding of peptides (13). There is some evidence to suggest that these proteins can function as molecular chaperons (11), with the ATPase being coupled to the folding of proteins and the assembly and disassembly of multisubunit structures (33, 37, 41). For example, the cytosolic hsc70 can dissociate clathrin from clathrin-coated vesicles in vitro in an ATP-dependent reaction (6). However, the large molar excess of hsc70 in growing cells relative to clathrin indicates that there are other functions for this protein. In yeast cells, members of the hsp70 family have been shown to be involved in the translocation of proteins into mitochondria and the endoplasmic reticulum (8, 9). In mammalian cells, there is evi-

Corresponding author. 2186

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The 70-kDa heat shock protein hsp7O and its constitutively expressed cognate, hsc7O, are abundant proteins implicated in a number of cellular processes. When a permeabilized cell system for examining the transport of proteins into the nucleus is depleted of hsc7O and hsp7O, either by affinity chromatography on ATP-agarose or with antibodies against these proteins, nuclear transport activity is lost. Full activity is restored by the addition of HeLa proteins that bind to ATP-agarose. hsc7O and hsp7O are the active factors, since activity is also fully restored by the addition of either recombinant hsc7O or hsp7O which has been bacterially expressed and highly purified. The restoration of activity is saturable. The transport system requires other cytosolic factors as well, including at least one protein that is sensitive to inactivation by N-ethylmaleimide, but neither hsc7O nor hsp7O is the sensitive protein.

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hsc7O AND hsp7O IN CYTOPLASMIC-NUCLEAR TRANSPORT

dence to suggest that cytosolic hsc70 is involved in the transport of proteins to lysosomes (7), to mitochondria (42), and, as shown here, to the nucleus.

placed in ice-cold transport buffer containing 3 mM ATP and 3 mM MgCI2. The addition of magnesium and ATP to the permeabilizing buffer results in lower background levels of hsp70 and hsc70. Digitonin is added to 40 ,tg/ml from a 20-mg/ml stock in dimethyl sulfoxide, and after 5 min, the coverslip is transferred to fresh ice-cold transport buffer for 5 min. The coverslip is placed face down on a microscope slide on top of a 40-,u drop of transport mixture (containing, in transport buffer, 1 mM ATP, 5 mM creatine phosphate, 20 U of creatine phosphokinase per ml, 6 ,g of TRITCnucleoplasmin [28] per ml, and 8 ,ul or the indicated amount of the HeLa cell extract that is described below). After incubation in a humid box at 30°C for 20 min, the cells are observed through a Zeiss Axiophot microscope and photographed with T-Max 400 film, using the same exposure time for all experiments. The negatives are digitized with an Apple computer and Color Imaging System Quick Scanner (Barneyscan Corp., Berkeley, Calif.), and the optical density of an area over the nucleus (excluding the nucleolus) of each cell is determined. Duplicate experiments are done for each data point, and measurements of about 20 nuclei from the two experiments are averaged. Each set of experiments includes as references samples with 0 and with 4 mg of HeLa cell extract per ml, which are defined as having nuclear localization of 0 and 1, respectively. To calculate nuclear localization, the average optical density over the nuclei of the zero reference is subtracted from the average optical density over the nuclei of the sample. This is then divided by the average optical density over the nuclei of the reference experiment done with 4 mg of HeLa extract per ml. A nuclear localization of 1 represents about a 20-fold-greater concentration of nucleoplasmin in the nucleus than in the cytoplasm. To prepare a HeLa cell cytosolic extract, 6 liters of HeLa cells growing in suspension is harvested at 6 x 105 cells per ml and washed twice with phosphate-buffered saline containing 50 mM glucose. The cell pellet (5 ml) is resuspended in 7.5 ml (1.5 times the volume of the cell pellet) of ice-cold water containing 1 mM phenylmethylsulfonyl fluoride and 1 ,ug each of aprotinin, leupeptin, and pepstatin per ml. After 15 min, the cells are homogenized by 10 strokes of a Dounce homogenizer, with cell lysis being monitored by phasecontrast microscopy. The extract is centrifuged at 1,000 x g for 10 min, and the supernatant is again centrifuged at 17,000 x g for 20 min. This supernatant is dialyzed against transport buffer and again centrifuged at 17,000 x g for 20 min and 100,000 x g for 30 min. The protein concentration is usually 20 mg/ml. The extract is stored in single-use portions at -800C. Depletion, inactivation, and reconstitution of extracts. A HeLa cell extract (1 ml) prepared as described above is depleted of ATP-binding proteins by passing it three times through an ATP-agarose column containing 0.25 ml of swollen ATP-agarose attached through the C-8 position (Sigma) that has been equilibrated with transport buffer. The HeLa cell cytosolic extract (40 pul) is depleted of hsc70 and hsp7o by incubation for 1 h at 40C with anti-C7-protein A-agarose and then pelleting of the complex. Anti-C7-protein A-agarose is formed by incubating 100 RI of antiserum with 50 pul of protein A-agarose (Sigma) for 1 h and then washing the sample three times with transport buffer. The inactivation of extract and modification of hsc70 and hsp70 with N-ethylmaleimide are done as previously described (2). Depleted extract (containing 160 pug of protein) is reconstituted by adding HeLa cell ATP-binding proteins or recombinant hsc70, hsp70, or cpn6o to give the indicated final

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MATERIALS AND METHODS Materials. The anti-hsp7o antiserum (gift from A. Frey, New York University Medical School) is directed against the carboxy-terminal 21 amino acids of hsp70 (16). The anti-C7 antiserum (gift from R. Reese, Agouron Institute, La Jolla, Calif.) is directed against a 28-kDa carboxy-terminal fragment of a Plasmodium falciparum 75-kDa surface protein (36) that is homologous with hsp70 and hsc70 (24, 36). The N27 and C92 anti-hsp7o/hsc7o antibodies (27) are from W. Welch (University of California, San Francisco). Nucleoplasmin is isolated from Xenopus eggs and conjugated with tetramethylrhodamine-,B-thiocyanate (TRITC) as previously described (28). When analyzed by nondenaturing gel electrophoresis in 5% polyacrylamide, a single band which moves at the same position in the presence or absence of 4 M urea is detected. When the preparation is examined by electron microscopy with 2% uranyl acetate negative staining, no aggregates are seen. The hsc70 clone was constructed by inserting rat hsc70 cDNA (15) between the NdeI and BamHI sites of a pET-11 expression vector. An NdeI site containing the AUG start codon of the hsc70 cDNA was generated by a polymerase chain reaction. The hsp70, also in a pET-11 expression vector, is a gift of R. Morimoto (Northwestern University). Both proteins are expressed and purified by the same protocol. Bacteria grown toA550 = 1.0 are induced by the addition of isopropyl-p-D-thiogalactopyranoside to 0.4 mM. After 30 min, rifampin is added to 25 ,ug/ml; the bacteria are grown for 2 h longer and then harvested by centrifugation. The bacteria are resuspended in 50 mM Tris-HCl (pH 8.3)-i mM EDTA-1 mg of lysozyme per ml to 1% of the volume of the original culture and disrupted by sonication. The extract is cleared by centrifugation at 17,000 x g for 20 min and at 100,000 x g for 30 min. The supernatant is applied to a hydroxylapatite column (13 by 50 mm) and developed with a 20 to 500 mM linear gradient of potassium phosphate (pH 7.6). The peak fractions (as determined by sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis) are applied to a QMA Accell (Waters) column (13 by 50 mm) and developed with a 20 to 400 mM NaCl linear gradient in 20 mM Tris-acetate (pH 7.6). The peak fractions are applied to a 1-ml ATP-agarose (Sigma) column, and after a wash with transport buffer, the proteins are eluted with 3 mM ATP in transport buffer. At this stage the proteins are better than 95% pure, as determined by SDS-polyacrylamide gel electrophoresis. Chinese hamster cpn6o, expressed in bacteria from cDNA cloned into a pET-11 vector (44), is a gift from N. Cowan (New York University Medical School). HeLa cell ATP-binding proteins are prepared by passing a HeLa cell cytosolic extract (see below) through an ionexchange column as described for the purification of hsp7o proteins (45) and passing the eluate through an ATP-agarose (linked through C-8; Sigma) column. The ATP-binding proteins are eluted with 3 mM ATP as described previously (45). In vitro assay for nuclear transport. HeLa cells grown on glass coverslips are gently blotted to remove excess medium, washed briefly with ice-cold transport buffer (20 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.3], 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol, 1 ,ug each of aprotinin, leupeptin, and pepstatin per ml), and

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concentration of added protein. The volume is then adjusted to 40 p.1 with transport buffer. RESULTS ATP-binding proteins are required for nuclear localization. Since the transport of proteins from the cytoplasm to the nucleus requires the hydrolysis of ATP (3, 5, 29, 35), we reasoned that it might be possible to use affinity chromatography on ATP-agarose to identify and purify factors required for nuclear transport. As an assay, we have used an in vitro

B

A

1.4 1.2

a 0

0

% 1.0

0

0.8

laU

0 0.6

0

z 0.4

z

a

0

N

C)

0 a

0.2 10

15

20

150 200 250 50 100 /rnl AIPD R-Ai-" P LL) VhKI A1r DninainJg rLoteisUI-ren

Extract (mg/ml) FIG. 2. Depletion and reconstitution of nuclear localization activity. (A) The ability of digitonin-permeabilized HeLa cells to transport TRITC-nucleoplasmin into the nucleus is dependent on factors present in a HeLa cell cytosolic extract (@), one or more of which are removed by passing the extract through an ATP-agarose affinity column (A) but not by a column of underivatized agarose (0). (B) The HeLa cell extract depleted by passage through an ATP-agarose column as in panel A is reconstituted at a protein concentration of 4 mg/ml with the indicated amount of HeLa cell ATP-binding proteins prepared as described in Materials and Methods. Nuclear localization is assayed as in Fig. 1, with the data being quantitated by densitometry of photographic negatives as described in Materials and Methods. Each data point shows the average and standard deviation of data collected in duplicate experiments from approximately 20 cells.

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FIG. 1. Nuclear localization in permeabilized HeLa cells complemented with transport buffer (A), a HeLa cell cytosolic extract containing 5 mg of protein per ml (B), the same HeLa cell extract depleted of ATP-binding proteins by passage through an ATPagarose column (C), and the extract in panel C supplemented with 250 jig of HeLa cell ATP-binding proteins per ml (D). Digitoninpermeabilized HeLa cells are incubated with the indicated buffer or extract, ATP, an ATP regeneration system, and TRITC-nucleoplasmin as described in Materials and Methods.

nuclear localization system based on the one developed by Adam et al. (1, 2), in which the import of exogenously added TRITC-nucleoplasmin into nuclei of permeabilized HeLa cells is measured. This system is specific for nuclear proteins: while nuclear proteins such as TRITC-nucleoplasmin are transported into the nucleus, other nonnuclear proteins such as TRITC-bovine serum albumin are excluded. In this assay, HeLa cells are permeabilized by digitonin, which not only permeabilizes the cells but also causes a loss of factors necessary for nuclear transport. These factors can be resupplied by incubating the permeabilized cells with a HeLa cell cytosolic extract, ATP, and an ATP regeneration system. We have developed a procedure by which a highly active HeLa cell cytosolic extract can be consistently obtained. The ability of this extract to reconstitute the nuclear localization activity of permeabilized HeLa cells is shown in Fig. 1. While permeabilized cells in the presence of transport buffer with no added cytosolic extract are very ineffective in transporting the TRITC-nucleoplasmin into the nucleus (Fig. 1A), the addition of cytosolic extract at a protein concentration of 5 mg/ml restores the ability of the permeabilized cells to transport the TRITC-nucleoplasmin into the nucleus (Fig. 1B). A concentration of nucleoplasmin in the nucleolus is also seen. It is not clear whether this nucleolar accumulation is due to a specific intranuclear transport pathway or merely the binding of nucleoplasmin, which is not a HeLa cell protein, to nucleolar components. Data from experiments with different concentrations of cytosolic extract are shown in quantitative form in Fig. 2A. Each data point represents the average and standard deviation of data collected in duplicate experiments from approximately 20 cells. For each titration, the data are collected from a single batch of cells seeded and grown on coverslips in the same dish. There is a somewhat greater variability between different batches of cells grown in separate dishes, and for this reason the data from each titration include, as positive control, an experiment done with 4 mg cytosolic extract per ml and, as a negative control, an experiment done with buffer. The data are normalized to these two controls on a scale of 0 to 1 as described in Materials and Methods. A nuclear localization of 1 represents about a 20-fold-greater concentration of

VOL. 12, 1992

hsc7O AND hsp7O IN CYTOPLASMIC-NUCLEAR TRANSPORT

A

B

1

2

3

C

1

2

3

A

1 2

3 .

B

2189

C

1.0

0

< 0.8 ;n

116 k 84 k -

58 k 48 k -

som rnWA

-aop

36 k 26 k -

nucleoplasmin in the nucleus than in the cytoplasm. All of the quantitative data reported in this paper are from experiments done with aliquots of the same preparation of cytosolic extract. Experiments done with other preparations of extract give, qualitatively, the same results. When the cytosolic extract is passed through an ATPagarose (covalently linked through the C-8 position of ATP) affinity column, its ability to complement nuclear localization in the permeabilized cell assay is greatly reduced (Fig. 1C and 2A). The same treatment with underivatized agarose, however, causes only a slight loss in activity (Fig. 2A). The activity of the depleted extract can be reconstituted by adding back the HeLa cell proteins that bind to ATP-agarose and are eluted by 3 mM ATP (Fig. 1D; results from a number of experiments done with differing amounts of added ATPbinding proteins being shown in quantitative form in Fig. 2B). From these experiments, we conclude that the inhibitory effect of ATP-agarose is caused by the sequestering of an essential component which binds to and can be eluted from ATP-agarose. The material eluted from ATP-agarose contains three major proteins of about 70 to 75 kDa (Fig. 3A, lane 1). About 1 mg of protein is obtained from 6 x 108 cells. Thus, these proteins are abundant, comprising approximately 1% of the total cellular protein. Their size, abundance, and purification properties suggest that they might be members of the hsp7O family of proteins. To further explore this possibility, the ATP-agarose-binding proteins are analyzed by immunoblotting with antibodies (described below) that recognize both hsp70 and hsc7O. As shown in Fig. 3, it is apparent that proteins recognized by these antibodies are major components of this fraction. Antibodies against hsp7O family members block nuclear localization. To test the possibility that the hsp70 proteins are the active components in the ATP-agarose-bound material, we have investigated the effects of several antibodies which recognize hsp70 family members on nuclear localization.

0.6

-

0.4

:3 z

0.2

0 0.2 0.4 0.6 0.8

0.2 0.4 0.6 0.8 Antiserum (Al)

0.2 0.4 0.6 0.8

FIG. 4. (A and B) Inhibition of nuclear localization by anti-hsp70 (A) or anti-C7 (B); (C) effect of a nonimmune serum which recognizes neither hsp70 nor hsc70 on immunoblots. The HeLa cell cytosolic extract shown in Fig. 2 (40 Ill containing 160 ,ug of protein) is incubated with the indicated amount of serum for 1 h at 4°C and then used in the nuclear localization assay of Fig. 2.

One antibody was prepared by immunizing rabbits with a 21-amino-acid C-terminal peptide of the human hsp70 (16). It recognizes both hsp70 and hsc7O, but with a preference for hsp7O (Fig. 3B). The addition of this antiserum to the HeLa cell cytosolic extract reduces the ability of the extract to complement permeabilized cells in the nuclear localization assay by about 80% (Fig. 4A). P. falciparum contains a highly antigenic 75-kDa surface protein that shares a great deal of homology with hsc70 and hsp70. Antibodies against the C-terminal third of this protein cross-react with both hsc70 and hsp70 (24, 36), but with a preference for hsc70 (Fig. 3C). As shown in Fig. 4B, these antibodies are highly effective at blocking nuclear localization. We have also examined the N21 and C92 monoclonal anti-hsp7o/hsc7o antibodies of Milarski et al. (27) but find them to be much less effective, possibly because of the nature of the epitopes that these antibodies recognize. The antibodies that block nuclear localization (Fig. 4) recognize the C-terminal regions of hsp70 and hsc70. When normal serum that does not react with 70-kDa proteins on immunoblots is used in these experiments, there is little, if any, effect on nuclear localization (Fig. 4C). Some sera from unimmunized animals detect 70-kDa proteins on Western immunoblots, presumably because of autoimmunity to these abundant proteins. HeLa cell extracts can be depleted of a factor that is essential for nuclear localization by incubating them with antibody-protein A-agarose (Fig. 5), while protein A-agarose complexed with immunoglobulin G from nonimmune animals has little or no effect on the activity of the extract. The activity of the immunoadsorbed extract can be reconstituted with the ATP-binding proteins that are described above, as shown in Fig. 5. Therefore, both ATP-agarose and immunoadsorption remove the same factor from the HeLa cell extract. These depletion experiments suggest that the protein recognized by the antibodies is a component of the cytosolic extract rather than the permeabilized cells. This conclusion is further substantiated by the finding that if permeabilized HeLa cells (as opposed to the HeLa cell extract) are incubated with antiserum and then complemented with untreated HeLa cell extract after a brief wash, there is no effect on nuclear localization (not shown). These results correlate with the loss of hsp7O and hsc70 from permeabilized cells, as detected by immunofluorescence. Cells permeabilized in the absence of ATP show a small amount of staining by antihsp7o, and when ATP is included in the permeabilization buffer (as described in Materials and Methods), the staining

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FIG. 3. Composition and immunoreactivity of ATP-binding proteins (lane 1), hsc7O (lane 2), and hsp7O (lane 3). The proteins are separated by SDS-polyacrylamide electrophoresis and then subjected to Coomassie blue staining (A), immunoblotting with antihsp7O serum (B), or immunoblotting with anti-C7 serum (C). Samples of the proteins used in Fig. 1, 2, 5, and 6 prepared as described in Materials and Methods are analyzed. The anti-hsp70 serum is directed against the carboxy-terminal 21 amino acids of hsp7O (16). The anti-C7 serum is directed against a 28-kDa carboxy-terminal fragment of a P. falciparum 75-kDa surface protein homologous with hsc70 and hsp7o (36).

a

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TABLE 1. N-ethylmaleimide sensitivity of cytosolic components of the transport system

1.2-

1.0

Nuclear

Treatment

0

Extract

/

,N 0.8 .90.6

3 0.4 0.2 100

150

250

200

Protein (jg/ml)

is reduced to background levels. ATP is known to facilitate the release of these proteins from nucleoli of heat-shocked cells (18). Recombinant hsp7O and hsc7O reconstitute nuclear localization activity. While the material that is eluted from ATPagarose by 3 mM ATP is enriched for heat shock proteins, it is possible that the activity resides in a minor component. To investigate this possibility and to identify the heat shock protein(s) involved, we tested the ability of bacterially expressed hsc70 and hsp7o to complement the depleted extracts. The proteins are expressed from cDNAs cloned into pET-11 expression vectors and then purified. The purities of the proteins used are shown in Fig. 3. The activity of HeLa cell extracts depleted either by ATP-agarose (Fig. 6A to C) or by antibody-protein A-agarose (Fig. 6D to F) is restored by either highly purified hsc70 (Fig. 6A and D) or

0

100

200

6.

F).

200

Protein (jsg/mI)

Reconstitution of depleted extracts with bacterially ex-

pressed recombinant hsc70 (A and D), and

100

200

Complementing

FIG.

The

NEMC

NEM NEM NEM

Untreated

extracts

used

in

panels

hsp70 A

to

(B and E), or cpn60 (C C

are

depleted

with

antibody-protein A agarose as in Fig. 5, and the extracts used in panels D to F are depleted with ATP-agarose as in Fig. 2. hsp70 and hsc70 are shown in Fig.

3,

cpn60

localization is assayed as in Fig. 2.

is described (44),

and

nuclear

hsp70

NEM

Untreated

localization' 1.06 0.95 0.08 0.06 -0.04 1.00

a Assayed as described in Materials and Methods by using 40 ,d of extract containing 160 ,ug of protein and, where indicated, 10 ,ug of hsc70 or hsp70. h HeLa cell cytosolic extract depleted of ATP-binding proteins as in Fig. 2. ' Treatment with 5 mM N-ethylmaleimide and quenching with 10 mM dithiothreitol (2). d Addition of dithiothreitol to 10 mM followed by treatment with 5 mM N-ethylmaleimide (2).

highly purified hsp70 (Fig. 6B and E). As judged from the amount of protein required to restore activity to the extract, hsc70 and hsp70 have nearly equal activities. Some members of the hsp70 family can act as chaperons (37, 41) or catalysts of protein folding and assembly. Since the mitochondrial chaperonin cpn60 catalyzes a similar type of reaction (23, 44), we tested the ability of bacterially expressed Chinese hamster cpn60 to complement the depleted HeLa cell extracts (Fig. 6C and F). While this same protein preparation is active in promoting the folding of ribulose bisphosphate carboxylase (44), it does not complement the depleted extracts.

hsp7O and hsc7O are not N-ethylmaleimide-sensitive factors. We know that hsp70 and hsc70 are not the only cytosolic factors required for nuclear localization, since permeabilized cells reconstituted with either of these proteins or with the HeLa cell ATP-binding proteins in the absence of depleted cytosolic extract are inactive. Nuclear localization requires factors that are sensitive to alkylation by N-ethylmaleimide (1, 2, 30). hsp70 and hsc70 are not these factors, since N-ethylmaleimide-modified hsp7o and hsc70 are fully active in reconstituting depleted extracts (Table 1). In another approach, we inactivated a HeLa cell extract by treating it with N-ethylmaleimide and then tested the ability of either hsc70 or hsp70 to restore activity. Both proteins are inert in this assay (Table 1), again showing that they are not the N-ethylmaleimide-sensitive factors. DISCUSSION In the experiments described above, we find that extracts from which hsc70 and hsp70 have been removed no longer support nuclear localization in an in vitro assay and that the addition of either hsc70 or hsp70 will restore this activity. Thus, the two proteins, which have very similar sequences and properties, appear to be able to function interchangeably. These proteins differ primarily in their levels of expression. The hsc70 protein is expressed at relatively constant levels, and the hsp70 protein is highly inducible. The synthesis of hsp70 increases not only as a consequence of environmental stresses but also during the cell cycle (26, 27), occurring concomitantly with an increase in the synthesis of nuclear proteins such as histones. The hsp70 mRNA levels increase 10- to 15-fold upon entry into the S phase and decline by late S phase and G2 (26). hsp70 is also expressed at high levels in transformed cells and is induced in cells that have been infected with DNA tumor viruses (46). Thus, in

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FIG. 5. Reconstitution of antibody-depleted extract with ATPbinding proteins. The HeLa cell cytosolic extract used for Fig. 2 is depleted by incubation with anti-C7-protein-A agarose (Materials and Methods). Portions of the depleted extract (40 ,ul containing 160 ,ug of protein) supplemented with the indicated amount of ATPbinding proteins (see Fig. 2) are used in nuclear localization assays as in Fig. 2.

100

Depletedb Depleted

Mockd 50

hsc70

VOL. 12, 1992

hsc70 AND hsp7O IN CYTOPLASMIC-NUCLEAR TRANSPORT

the simian virus 40 T antigen (39), the polyomavirus middle T antigen (31), and the v-rel gene product (20). Also, during cellular stress, hsc70 and hsp7O accumulate in the nucleolus, presumably because of associations with nucleolar proteins. hsc70 has also been shown to be associated with the cytoskeleton and microtubules (15). The hsc70 and hsp7O proteins are present in relatively large amounts, particularly in rapidly proliferating cells. Such quantities are entirely consistent with a role in nuclear protein transport, given the amounts of these proteins that are synthesized during S phase, the number of ribosomal proteins that must be directed to the nucleolus for the assembly of ribosomes, and the quantity of proteins that enter newly formed nuclei following mitosis. On the basis of the evidence presented here, we suggest that hsc70 and hsp70, the most abundant members of the hsp70 family, are involved in directing proteins to the largest organelle, the nucleus. ACKNOWLEDGMENTS We thank W. Welch, 0. Bensaude, R. Reese, A. Frey, and R. Li for antibodies, N. Cowan and R. Morimoto for clones, M. Nachbar for the use of the digitizing equipment, and all for discussion. This work was supported by the American Cancer Society. REFERENCES 1. Adam, S. A., and L. Gerace. 1991. Cytosolic proteins that specifically bind nuclear localization signals are receptors for nuclear import. Cell 66:837-847. 2. Adam, S. A., R. S. Marr, and L. Gerace. 1990. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111:807-816. 3. Akey, C. W., and D. S. Goldfarb. 1989. Protein import through the nuclear pore complex is a multistep process. J. Cell Biol. 109:971-982. 4. Beckmann, R. P., L. A. Mizzen, and W. J. Welch. 1990. Interaction of hsp70 with newly synthesized proteins: implications for protein folding and assembly. Science 248:850-854. 5. Breeuwer, M., and D. S. Goldfarb. 1990. Facilitated nuclear transport of histone Hi and other small nucleophilic proteins. Cell 60:999-1008. 6. Chappell, T. G., W. J. Welch, D. M. Schlossman, K. B. Palter, M. J. Schlesinger, and J. E. Rothman. 1986. Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell 45:3-13. 7. Chiang, H.-L., S. R. Terlecky, C. P. Plant, and J. F. Dice. 1989. A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 24:382-385. 8. Chirico, W. J., M. G. Waters, and G. Blobel. 1988. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature (London) 332:805-810. 9. Deshaies, R. J., B. D. Koch, M. Werner-Washburne, E. A. Craig, and R. Schekman. 1988. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature (London) 332:800-805. 10. Dingwall, C., S. V. Sharnick, and R. A. Laskey. 1982. A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell 30:449-458. 11. Ellis, R. J., S. M. van der Vies, and S. M. Hemmingsen. 1989. The molecular chaperone concept. Biochem. Soc. Symp. 55: 145-153. 12. Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. A complex of nuclear pore proteins required for pore function. J. Cell Biol. 114:169-183. 13. Flynn, G. C., T. G. Chappell, and J. E. Rothman. 1989. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science 245:385-390. 14. Garcia-Bustos, J., J. Heitman, and M. N. Hall. 1991. Nuclear protein localization. Biochim. Biophys. Acta 1071:83-101. 15. Green, L. A. D., and R. K. H. Liem. 1989. 3-Internexin is a

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the absence of cellular stresses, the expression of hsp70 is correlated with the requirement for the transport of proteins to the nucleus. The cellular location of hsc70 and hsp70 is also cell cycle dependent, with the proteins being localized in the nucleus and nucleolus during S phase and distributed throughout the cell during the rest of the cell cycle (26, 27). Mandell and Feldherr (22) have shown that two 70-kDa Xenopus proteins that are members of the hsp7o family recycle across the nuclear membrane of Xenopus oocytes and that hsc70 also does this when microinjected into Xenopus oocytes. These properties are, as Mandel and Feldherr have pointed out, consistent with hsc7O functioning as a carrier that transport nuclear proteins into the nucleus. While we have presented evidence to show that hsc70 and hsp70 are involved in the transport of proteins to the nucleus, there is evidence that hsc70 may be involved in the transport of proteins to other organelles as well. A role for hsc70 in the transport of proteins to lysozomes has been suggested on the bases of its association with the KFERQ peptide of RNase A, which is essential for enhanced degradation during serum starvation (7). hsc70 has also been shown to be present in a 200- to 250-kDa complex that is required for the import of proteins into the mitochondria, although a direct role for hsc70 in mitochondrial protein import has not been demonstrated (42). As with nuclear localization, mitochondrial protein targeting is sensitive to alkylation by N-ethylmaleimide, but the sensitive factor is neither hsc70 nor hsp70 (42). Other members of the hsp7O family have been shown to be involved in the transport of proteins to various organelles (8, 9, 33), and it is possible that these proteins share analogous mechanisms of action. hsp70 and hsc70 may also facilitate protein folding and assembly, acting as chaperons (37, 41) as, for example, in the uncoating of clathrin-coated vesicles. The possibility that hsc7O and hsp70 function indirectly by using this activity to dissociate the 150-kDa nucleoplasmin pentamer into monomers that then enter the nucleus can be discounted. The nucleoplasmin pentamer is a remarkably stable structure that does not dissociate during SDS-polyacrylamide gel electrophoresis. Moreover, it has been demonstrated that nucleoplasmin enters the nucleus as a pentamer without dissociating into monomers (10) and may enter when bound to colloidal gold particles (14, 43). We can also discount the possibility that nucleoplasmin aggregates are being dissociated by hsc70 or hsp70, since neither nondenaturing 5% polyacrylamide gel electrophoresis nor electron microscopy of the TRITC-nucleoplasmin shows the presence of aggregates in the TRITC-nucleoplasmin preparation. hsc70 contains a weak ATPase activity that is stimulated by the addition of peptides (13). The binding of peptides and the hydrolysis of ATP appear to be thermodynamically linked, and this activity probably lies at the heart of how the protein functions. Both the chaperon and transport functions involve the ATP-dependent binding and release of a protein. It is possible, therefore, that both functions are catalyzed by hsc70 and hsp70, with transport being a vectorial process. While a folding or chaperon activity may be present, it is probably not essential for the transport of all nuclear proteins, since a number of completely folded native proteins as well as nuclear protein-coated gold particles are capable of being transported into the nucleus (5, 14, 19, 29, 35, 43). hsc70 and hsp70 have been shown to associate with a number of proteins, particularly those that are newly synthesized (4). Only a few of these proteins have been identified, and these are mainly nuclear proteins such as p53 (16),

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