Purification of the Major Mammalian Heat Shock Proteins*

The major mammalian heat shock or “stress” pro- teins (molecular masses of 90,000, 72,000, and 73,000 daltons) have been purified from stressed HeLa cells. The 90,000-dalton protein co-purified with small amounts of a 100,000-dalton protein which was identi-fied as one of the other stress proteins in these cells. The 72,000- and 73,000-dalton proteins co-purified throughout the fractionation scheme, apparently as a mixture of monomeric forms of the two proteins. From sedimentation velocity and gel filtration analysis, it was found that the 90,000/100,000-dalton protein mix- ture had a Stokes radius of 69A and a 8 2 0 , ~ value of 5.8 while the 72,000/73,000-dalton protein mixture had a Stokes radius of 42.6A and a szo.w value of 4.3. The purified proteins migrated identically in two-dimen-sional gel electrophoretograms with their counterparts from total cell lysates of [35S]methionine-labeled stressed HeLa cells. Peptide mapping experiments indicated that the 72,000- and 73,000-dalton proteins con- tained common peptides while the 90,000- and 100,000-dalton proteins appeared to be distinct. Amino acid analysis of the 90,000- and a mixture of the 72,000/ 73,000-dalton proteins showed that both contained rel-atively high amounts of Asp/Asn and Glu/Gln. Eukaryotic and prokaryotic cells, when confronted with environmental insults, elicit a common and seemingly highly conserved response: the stress response. A diverse collection of treatments including exposure to drugs (l), amino acid analogues (1-4), transition series metals

elevated, nonphysiological temperatures (classically referred to as heat shock) (Ref. 6 and reviewed in Ref. 7) give rise to the "stress response." Some of the mechanistic and phenomenological aspects of the stress response in a variety of organisms have been analyzed (1,(7)(8)(9)(10).',' Briefly, once activated in eukaryotes, the stress response results in the induction of a limited number of proteins, the stress proteins, which eventually become the major polypeptide products of the stressed cell both in terms of their synthesis and accumulation. All of the stress proteins, with some minor exceptions, appear to be synthesized in normal tissue culture cells although at lower rates than in the stressed cell (ll).'" Increases in the synthesis (and in some cases, the amounts) of these proteins in the stressed cell appear to result from the near exclusive synthesis of mRNAs coding for these proteins as well as the reduced * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement" in accordance with 18  translation of pre-existing mRNAs coding for other cellular polypeptides (Refs. 12-17 and reviewed in Ref. 7). The mechanisms by which such transcriptional and translational controls are exerted in the stressed cell are not well understood.
The cellular response to stress appears to be well conserved in both vertebrate and invertebrate cells. The stress proteins induced in a number of different cell types by a variety of inducing agents have similar electrophoretic mobilities in onedimensional SDS4-polyacrylamide gels. For example, HeLa cells placed under stress in either of three ways, 1) growth in medium containing an amino acid analogue of proline, AzC; 2) growth in medium containing Zn'+; or 3) growth at elevated temperatures, i.e. heat shock, accumulate significant amounts of proteins with molecular masses of 110,000, 100,000, 90,000, 80,000, 73,000, and 72,000 daltons (8). In addition, in such stressed HeLa cells, synthesis of the normal complement of other cellular polypeptides is dramatically reduced.
Although it is known which proteins are induced during the stress response, their location and function in the cell have not been fully elucidated. There is evidence that induction of the stress response confers a degree of protection against subsequent stress situations and that such a protection is contingent upon the prior synthesis of the stress proteins (18)(19)(20). Furthermore, with regard to their intracellular location, there are reports showing that in insect cells some of the stress proteins appear to migrate to the nucleus shortly after their synthesis in the cytoplasm (21)(22)(23)(24)(25).
As a first step in the analysis of the function and intracellular location of the mammalian stress proteins, we report here the purification of three of the six stress proteins produced in HeLa cells. This purification scheme has been subsequently employed in the purification of the corresponding proteins present in normal unstressed cells. Acquisition of both the purified proteins (as well as the appropriate antibodies directed against them) should aid in the determination of the location and function(s) of the stress proteins both in normal and in stressed cells and thereby facilitate and expedite a more complete understanding of the stress response.

EXPERIMENTAL PROCEDURES
Growth of Cells and Induction of the Stress Response-Two liters of HeLa cells, seeded at 5 X IO5 cells/ml, were grown in suspension in F-13 spinners medium (Gibco) supplemented with 5% horse serum. The stress response was initiated 24 h later by one of three methods: (a) addition of ~-azetidine-2-carboxylic acid (Calbiochem) (final concentration, 5 mM) to the culture medium; (b) addition of ZnCl:! (final concentration, 0.25 mM); or ( c ) heating the medium to 42 "C. Between 8 and 16 h later (the time depending upon the treatment used for the induction), the cells were harvested and prepared for protein purification as described under "Results." Column Chromatography and Buffers-Whatman DEAE-cellulose (DE52 and DE53) was purchased from Reeve Angel and Co. Sepharose GB-CL and Sephacryl S-300 were obtained from Pharmacia Fine Chemicals. Hydroxylapatite was purchased from Bio-Rad. Salt The abbreviations used are: SDS, sodium dodecyl sulfate; AzC: ~-azetidine-2-carboxylic acid; DME, Dulbecco's modified Eagle's medium; 72 kd, 73 kd proteins, etc., proteins of 72 and 73 kilodaltons.

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concentrations in column effluents were measured with the use of a Radiometer conductivity meter. Buffer b (see below) gave a reading of 1.9 millisemens. Absorbance a t 280 nm of the column effluents was measured with a Varian 634 spectrophotometer.
Standards used to prepare a calibration curve for molecular weight determination by gel filtration were purchased from Pharmacia Fine Chemicals and included blue dextran ( M , = 2,000,000), ovalbumin ( M , = 43,000 and Stokes radius of 30.5A). and catalase ( M , = 240,000 and Stokes radius of 52.2A).
Two-dimensional Isoelectric Focusing a n d SDS-Polvacrylamide Gel Electrophoresis-The analysis was done as described by Garrels (29). Gels were stained with Coomassie blue as described above. Fluorography was done as described by Garrels (29).
[:'"S]Methionine-labeling of Normal and Stressed Cells-HeLa cells (-1 X 10" cells/dish) on 35-mm plastic dishes (Falcon) were grown in DME containing 2% calf serum. Cells were stressed by the addition of 5 mM AzC or 0.25 mM ZnCl? to the medium, or by growth in normal medium a t 42 "C. For labeling, the medium was removed, the cells were washed with DME lacking methionine, and then were labeled in methionine-free DME supplemented with [:'"S]methionine and 2% dialyzed calf serum under the appropriate stress condition. Following the labeling period, the medium was removed, the cells grown in suspension culture, were incubated a t 42 "C for 6 h in F-13 spinners medium supplemented with 5% horse serum. The cells were harvested, washed several times with phosphate-buffered saline, and then quickly washed with hypotonic medium (20 mM Tris-acetate (pH 7.5), 50 mM NaCI, 0.1 mM EDTA, 15 mM 2-mercaptoethanol). After collection by centrifugation, the cells were lysed by Dounce homogenization in cold hypotonic lysis buffer (10 mM Tris-acetate (pH 7.5), 10 mM NaCI, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride). The cell lysate was adjusted to 0.25 M sucrose, 1 mM MgCl? and centrifuged a t 1,000 X g (average) for 10 min a t 4 "C. The 1,000 X g supernatant was then centrifuged a t 100,000 X g (average) for 1 h a t 4 "C. Equal fractions of the pellets and supernatants were then analyzed on a 10% SDS-polyacrylamide gel. Molecular mass markers (lane A ) were, in descending order, 200,000, 130,000, 94,000, 68,000. 43,000,30,000, and 21,000 daltons. The 100,W. 80.73, and 72 kd stress proteins are indicated by hash marks to the left. Lane B, Douncehomogenized cell lysate (0.015% of total). Lane C, 1,000 X g pellet (0.03% of total). Lane D, 1,000 X g supernatant (0.03% of total). Lane E , 100,000 X g pellet (0.06% of total). Lane F, 100,000 X g supernatant (0.06% of total). were washed with cold phosphate-buffered saline, and the proteins were solubilized by the addition of SDS-gel electrophoresis sample buffer. The samples were boiled for 5 min and then passed through a 28-gauge needle three times to shear DNA.
Amino Acid Composition-The purified 72/73 kd mixture obtained after gel filtration over Sepharose GB-CL, and the 90 kd protein obtained after gel filtration over Sephacryl S-300 were solubilized with SDS-gel electrophoresis sample buffer and chromatographed on 7.5% SDS-polyacrylamide gels. The proteins were visualized by Coomassie blue staining and the protein bands were excised from the gel. The proteins were then electroeluted from the gel slices as described by Welch et al. (30), dialyzed extensively against 30 mM NH.,HCO:I, 0.01% SDS, and lyophilized. The lyophilized proteins were then resuspended in 10% acetic acid and passed over a G-25 column to remove all salts and any residual glycine from the SDS-gel electrophoresis running buffer. Fractions from the G-25 column were lyophilized, oxidized with performic acid, and hydrolyzed in 6 N HCI at 100 "C for 24 h in vacuo. Amino acid compositions were determined using a Beckman 119CL automated analyzer.

Fraction no.
Peptide Maps-The purified 72/73 kd mixture obtained after gel filtration over Sepharose 6B-CL and the purified 90/100 kd mixture obtained after gel filtration over Sephacryl S-300 were solubilized by the addition of SDS-gel electrophoresis sample buffer, and the proteins were chromatographed on a 7.5% SDS-polyacrylamide gel, and visualized by Coomassie blue staining. Individual 72, 73, 90, and 100 kd proteins were carefully excised from the gels, electroeluted out of the gel slices, dialyzed extensively against 20 mM sodium phosphate (pH 7.4), 0.01% SDS, and lyophilized. The individual proteins were resuspended in HIO and iodinated with N a 9 using the chloramine-T method (31). Following their iodination, the proteins were passed over a G-25 column, and the peak fractions were combined, solubilized in SDS-gel electrophoresis sample buffer, and chromatographed on a 108 SDS-polyacrylamide gel. T h e gel was dried and the protein was visualized by autoradiography.
For peptide mapping, the individual proteins were excised from the dried gel and mapped essentially as described by Cleveland et al. (32).

RESULTS
The purification procedures described below have been applied to cells grown in normal medium and cells stressed by either growth in medium containing 5 mM AzC, growth in medium containing 0.25 mM ZnCl?, or growth at 42 "C in normal medium. In general, except for the absolute amounts of the stress proteins induced by these various treatments, essentially identical results were obtained with respect to the purification, gel filtration, and sedimentation characteristics of the proteins. However, for the sake of brevity, the results of the fractionation of HeLa cells stressed by growth in the presence of AzC are presented.
The addition of AzC to the growth medium results in the elevated synthesis of a small number of polypeptides concomitant with the decreased synthesis of most other cellular polypeptides (

Initial Steps in the Purification of the Stress Proteins-In
order to facilitate the purification of the stress proteins, we first examined their relative subcellular distribution, using a simple fractionation procedure based on velocity sedimenta- tion. Following their incubation at 42 "C (i.e. heat shock) for 8 h, HeLa cells were collected by centrifugation and washed three times with cold phosphate-buffered saline. The cells were swollen by washing once in cold hypotonic medium (50 mM NaCl, 20 mM Tris-acetate (pH 7.5), 0.1 mM EDTA, 15 mM 2-mercaptoethanol) and after collection by centrifugation lysed by Dounce homogenization in cold h-ypotonic lysis buffer (10 mM Tris-acetate (pH 7.5), 10 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride. MgC12 was added to a final concentration of 1 mM and the cell lysate was then fractionated by two velocity sedimentation centrifugations. The protein composition during each step of the fractionation was determined by SDS-gel electrophoresis (Fig. 2). The identification of the stress proteins in each of the crude fractions was confirmed by two-dimensional gel electrophoresis (not shown). The Dounce-homogenized cell lysate (Fig. 2, lane B

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(average) for 10 min at 4 "C. A large pellet was obtained and contained appreciable amounts of all six of the stress proteins (Fig. 2, lane C ) . In relative terms, little of the 90 and the 100 kd stress proteins were found in this low speed pellet. The supernatant, however, contained most of the 90 kd protein as well as considerable amounts of 72,73,80, and 100 kd proteins for 16 h in culture medium supplemented with 5% horse serum and 5 mM AzC. Suspension cells were used to ensure adequate starting amounts of the stress proteins. The cells were harvested by centrifugation a t 1000 X g for 15 min in a Sorvall GSA rotor. The cells were washed twice with cold phosphatebuffered saline lacking Ca" and M$+ and once with cold hypotonic buffer (as above). The cellular pellet (approximately 3-4 g of packed cells) was then resuspended in cold hypotonic lysis buffer (as above). After swelling on ice for 20 min, the cells were lysed by Dounce homogenization using a tight fitting pestle. The homogenate (analyzed by SDS-gel electrophoresis, Fig. 2, lane 2 ) was centrifuged at 20,000 X g (maximum) for 15 min in a Sorvall SS-34 rotor at 4 "C. A large pellet consisting of mitochondria, nuclei, and unbroken cells was obtained. The supernatant was separated from the pellet and diluted with Buffer b until the conductivity approached that of the conductivity of Buffer b. This solution (Fig. 3, lane 4 ) was applied to a column packed with DE52 (1.2 x 20 cm) equilibrated in Buffer b and the column subsequently was washed with buffer until the A2R0 returned to a base-line value. A number of polypeptides did not bind to the column (Fig. 3, lane 5). Those proteins which did bind to the column were eluted with a linear gradient of NaCl ( Fig. 3; the beginning of the gradient is indicated by the arrow). Four principal peaks of optical density were found to elute from the column, two of which contained substantial amounts of three of the stress proteins. The first peak (fractions 20-34) was enriched in the 72 and 73 kd stress proteins, although both proteins were seen to extend well throughout the gradient. The third peak (fractions 35-45) contained substantial amounts of 90 kd and some 100 kd as well. It is important to note here that the 100 kd protein present in fractions 35-45 represents only a portion of the total present in the stressed cell. As described above, our subcellular fractionation results, as well as other recent studies, have shown that some of the 100 kd protein remains in the pellet following the 20,000 X g centrifugation of the Dounce-homogenized cell lysate5 (see "Discussion"). The two other stress proteins that are indicated in Fig. 1, the 110 and 80 kd proteins, were not unambiguously detected in this elution profie. As was shown in Fig. 2, a significant portion of the 80 kd protein remains with the pellet following the 1,000 X g centrifugation. For their further purification, the two peak fractions containing the 72 and 73 kd proteins and the 90 and 100 kd proteins were pooled separately and dialyzed extensively against Buffer c. w. J. Welch and J. R. Feramisco, manuscript in preparation.

Purification of the 90 kd Stress Protein-
The pooled fractions containing the majority of the 90 and 100 kd proteins were clarified by centrifugation a t 10,000 x g for 10 min at 4 "C following dialysis against Buffer c. The supernatant was applied to a hydroxylapatite column (1.2 X 10 cm) equilibrated in Buffer c. The column was washed with buffer until the A , returned to a base-line value and the proteins were eluted with a linear gradient of potassium phosphate (pH 7.5). One major peak of optical density was observed to elute from the column (Fig. 4). This peak, composed of fractions 45-56, contained the vast majority of the 90 kd protein. These fractions, containing most of the 90 kd protein and some of the 100 kd protein, were pooled and concentrated by negative pressure dialysis against Buffer b using a Micro-Pro-Dicon concentrator (Bio-Molecular Dynamics, Beaverton, OR).
The concentrated proteins, in a volume of approximately 1.5 ml, were chromatographed on a Sephacryl S-300 column (1.2 X 100 cm) developed in Buffer b. A single peak of optical density (fractions 42-49) eluted from the column (Fig. 5). The 90 kd protein, as well as some 100 kd, eluted together in these fractions with minimal contamination from other proteins.  tons stress protein.

Purification of the 72 and 73 kd Stress Proteins-The
pooled fractions from the DE52 column containing the 72 and 73 kd proteins were clarified after dialysis against Buffer c and applied to a hydroxylapatite column (1.2 X 10 cm) equilibrated in Buffer c. The column was washed with Buffer c until the AZRO " , , , revealed a base-line value. Proteins were then eluted with a linear gradient of potassium phosphate (pH 7.5) giving rise to two major and two minor peaks of optical density, the latter comprising the bulk of both the 72 and 73 kd proteins (Fig. 6). As in the initial step of the purification using DE52 chromatography, both the 72 and 73 kd proteins displayed a wide profile across the gradient. In addition, they again appeared to elute together in apparently an equimolar mixture. Fractions 54-70, containing the majority of the 72 and 73 kd proteins, were pooled and concentrated by negative pressure dialysis against Buffer b.
Once concentrated to a volume of approximately 2 ml, the solution containing the 72 and 73 kd proteins was applied to a Sephacryl S-300 column (1.2 X 100 cm) and the column was developed in Buffer b. As can be seen in Fig. 7, a number of proteins including 72 and 73 kd were included in the column. Those fractions containing most of the 72 and 73 kd proteins (fractions 35-45) were pooled conservatively to minimize contamination from the other polypeptides present. The combined fractions containing 72 and 73 kd were then applied directly to a DE53 column (1.2 X 10 cm) equilibrated in Buffer b. Because of the slightly different performance characteristics of this resin compared to DE52, this step improved the purity of the 72 and 73 kd proteins. The proteins were eluted from the column with a linear gradient of NaCl (Fig. 8). A single peak of optical density containing most, if not all, of the 72 and 73 kd proteins was detected. Fractions 30-43, the peak fractions, were pooled and concentrated by negative pressure dialysis against Buffer b.
As a final purification step, the concentrated fractions containing both the 72 and 73 kd proteins can be applied to a Sepharose 6B column (2.5 X 90 cm) equilibrated in Buffer b (not shown). This step provides little further purification of the proteins but does provide some side fractions that contain homogeneous 72 and 73 kd proteins. In the final pooled preparation, small amounts of contaminating proteins with molecular masses of approximately 100,000,35,000, and 32,000 daltons were detected on our SDS-polyacrylamide gels. We do not know whether these latter polypeptides are actually associated with the 72 and 73 kd proteins or merely represent minor contaminants. A yield of approximately 2-3 mg of the 72/73 kd mixture was obtained from 3-4g of cells (wet weight).  1.4-ml fractions (80 total) were collected and the AI,, (0) and conductivity (millisemens) (0) were determined. The flow rate was 20 ml/h. Shown helow is a Coomassie blue-stained 10% SDS-polyacrylamide slab gel of a portion of every third fraction. patterns on two-dimensional gels with the pattern of ['"S] methionine-labeled polypeptides synthesized in HeLa cells exposed to AzC was undertaken. The [""Slmethionine-labeled proteins synthesized in AzC-treated HeLa cells were combined with either the purified unlabeled 72/73 kd or with the unlabeled 90/100 kd proteins and the mixtures were analyzed by two-dimensional gel electrophoresis (Fig. 9). The gels were first stained with Coomassie blue to identify the unlabeled purified 90 and 100 kd proteins (Fig. 9B) as well as the 72 and 73 kd proteins (C). Subsequently, the two-dimensional gels were fluorographed to reveal the radiolabeled in viuo-synthesized stress proteins ( A ) . Superimposition of the fluorograph upon the Coomassie blue-stained gel demonstrated that the purified 72, 73, 90, and 100 kd proteins co-migrated with the corresponding [:'5S]methionine-labeled 72, 73, 90, and 100 kd proteins made in viuo. This then indicates that the proteins purified are the stress-induced 72, 73, and 90 kd proteins and that the proteins are not significantly altered during their purification.

Analysis of the Purified 72, 73, and 90 kd Stress Proteins by Two-dimensional Gel Electrophoresis-To
Physical Properties of the Stress Proteins-To determine the native molecular mass of the purified stress proteins, we measured both their Stokes radii by gel filtration and their sedimentation coefficients by density gradient centrifugation as described by Martin and Ames (28). Both the 72 and 73 kd proteins and the 90 and 100 kd proteins co-sedimented through 5-20% (w/v) sucrose gradients in a manner similar to their co-purification during column chromatography. Hence, the Stokes radii and sedimentation coefficients presented in Table I refer to the 72/73 kd mixture and the 90/100 kd mixture. Whether these proteins actually exist as complexes in vivo, however, is not clear at this time. A Stokes radius of 42.6A and a sedimentation coefficient ( sno,,,.) of 4.2 were found for the 72 and 73 kd mixture. Similarly, the 90/100 kd mixture displayed a Stokes radius of 69A and a sedimentation coefficient ( S~~~. , J of 5.8. Using these values, the native molecular mass for the proteins was determined using the method of Siegal and Monty (33). A native molecular mass of 73,800 daltons was obtained for the 72/73 kd mixture and a molecular    mass of 165,000 daltons for the 90/100 kd mixture. A summary of this data as well as the apparent molecular masses of the SDS-denatured proteins and their major isoelectric points as determined by isoelectric focusing (8) are presented in Table  I.
The amino acid composition of the 72/73 kd mixture and the 90 kd protein was determined next. The proteins were further purified by SDS-polyacrylamide gel electrophoresis, eluted from the gel, dialyzed extensively to remove salts and other impurities, and finally oxidized by treatment with performic acid. The amino acid composition of the 72/73 kd mixture and the 90 kd protein, as determined on a Beckman 119 CL automated analyzer, is presented in Table 11. A comparison of the composition determined for the 72 and 73 kd proteins agrees well with the predicted amino acid composition of the analogous 70 kd protein from Drosophila melanogasfer (as deduced from the nucleotide sequence of the gene (40)).
In light of the observations that the 90 kd and some of the 100 kd, as well as the 72 and 73 kd, proteins co-purified during column chromatography and co-sedimented during equilibrium sucrose sedimentation, the relationship of the proteins by one-dimensional peptide mapping was examined. Each individual protein was further purified by SDS-polyacrylamide gel electrophoresis, the proteins were eluted from the gels and iodinated, and peptide maps were generated via the method of Cleveland et al. (32) (see "Experimental Procedures" for details). Analysis of the 90 and 100 kd proteins, digested with varying amounts of Staphylococcus aureus V8 protease and examined on a 15% SDS-polyacrylamide gel, revealed no obvious similarities (Fig. 10). A similar analysis of the 72 and 73 kd proteins, however, demonstrated a considerable amount of homo!ogy between the two proteins ( Fig.  11). (Analysis of the tryptic peptides of 72 and 73 kd proteins by high pressure liquid chromatography revealed a similar homology between the two proteins.') These results appear consistent with those of Hightower and White (4) who showed that the analogous 72 and 73 kd stress-induced proteins present in cultured rat embryo cells are related polypeptides as determined by one-dimensional peptide mapping. It would appear then that the 90 and 100 kd proteins are not related to one another while the 72 and 73 kd proteins are similar but not identical polypeptides.

DISCUSSION
The phenomenon of stress (or heat shock) in eukaryotes involves a specific set of coordinate changes within the cell. While considerable work has focused on the genes induced and their corresonding mRNAs synthesized during the stress response, only recently has much attention been paid to the stress proteins themselves in terms of their structure, intracellular location, and function. To these ends then, we have begun purifying the six major HeLa stress proteins and here we report the purification of three of the six. A schematic outline of this purification is presented in Fig. 12.
Growth of suspension HeLa cells for periods of 4 to 16 h in medium containing 5 mM AzC, an amino acid analogue of proline, results in the induction of six proteins with apparent molecular masses of 72, 73,80,90, 100, and 110 kilodaltons as determined by their migration in one-and two-dimensional polyacrylamide gels (e.g. Ref. 8). Similar induction of these proteins is observed in cells grown in medium containing 0.25 mM ZnClz or grown under heat shock conditions. The enhanced synthesis of the same six proteins also occurs in chick embryo fibroblasts, in gerbil fibroma cells, and in baby hamster kidney cells grown under stress (data not shown). The similarities in the molecular masses of the stress proteins induced in these different cell lines indicates that the response to altered growth conditions in vitro is apparently well conserved.
Fractionation by differential sedimentation of HeLa cells grown under heat shock revealed that most of the 90 kilodaltons and about one-half of the 72 and 73 kd stress proteins partition into the soluble phase following lysis of the cells in low ionic strength buffer. Conversely, it was found that the particulate fraction after centrifugation of the Dounce-homogenized cell lysate was enriched in both the 80 and 100 kd stress proteins. Some portion of both the 80 and 100 kd proteins, however, was also present in the supernatants following the low and high speed centrifugations. The presence of the 100 kd protein in the particulate fraction is consistent with our recent observation that this protein is located, as determined by immunofluorescence, in or near the Golgi apparatus in a number of different cell types so far examined? The observation that some of the 72 and 73 kd proteins fractionate with the low speed pellet is consistent with the findings of others (25,39) that a portion of the apparently analogous 70 kd heat shock proteins of D. melanogaster cells appear to be present in the nucleus.
Because of the interest in determining the identity, intracellular location, and function of the stress proteins, it seems appropriate here to discuss what is presently known about these proteins. First, it seems clear that all of the stressinduced proteins (with a few minor exceptions) are present in tissues and in uninduced "normal" tissue culture cells (4, 11). While the 72, 80, 100, and 110 kd proteins are present in apparently low amounts, the 73 and 90 kd proteins appear as prominent proteins in a variety of different cell types grown in The effect of growing cells in tissue culture, however, may itself be a stressful situation and thereby result in a slight induction of these proteins as compared to the in vivo tissue.
Nevertheless, it would appear reasonable to assume that all of these proteins, in addition to serving in the stress response, With regard to the 90 kd protein, a number of laboratories have observed that a portion of the transforming protein of Rous sarcoma virus, pp60"", appears to exist as part of a complex with both a 50 kd protein and the 90 kd stress protein (34-36). More recently, Adkins et al. (37) have shown that a fraction of the putative transforming proteins of PRCll avian sarcoma virus, p105 and p110, similarly exist in a complex with a 50 kd protein and the 90 kd stress protein. In light of these reports detailing the associations of 90 kd with other cellular proteins, the observation that small amounts of the 100 kd stress protein (which is mainly associated with the Golgi apparatus6) co-purify with the 90 kd protein is most intriguing. The significance of these findings concerning the existence of various complexes containing the 90 kd protein, however, is still unknown. Interestingly, it also has been reported that the 90 kd protein in crude extracts of chicken cells appears to have, upon gel filtration, an M, = 560,000 (11). Although we find a lower value for the native molecular mass of the purified 90 kd protein (165,000 daltons), the higher value reported may reflect an association of the 90 kd protein with other proteins in such crude extracts.
Concerning the subcellular location of the stress proteins, a number of groups have observed a migration of the newly synthesized stress proteins to the nucleus in stressed Drosophila cells (21)(22)(23)(24)(25). In most of these studies, proteins with a molecular mass of approximately 70,000 daltons were found W. J. Welch, J. I. Garrels, and J. R. Feramisco, manuscript in preparation.
to be enriched in the nuclear region. Additionally, Wang et al. (38) have reported that two apparent heat shock proteins of approximately 68,000 daltons are associated with the cytoskeletal network in both avian and mammalian cells. The authors also reported that these proteins are methylated in both normal and heat-shocked cells. The possible relationship of these proteins to the 72/73 kd stress proteins described here is currently being examined.
Purification of the remaining three HeLa stress proteins, 80, 100, and 110 kd, is in progress in our laboratory. We have recently made antibodies in rabbits against each of the six HeLa stress proteins and are currently examining their intracellular location by immunofluorescence. With both the purified proteins as well as their corresponding antibodies available, it may be possible to determine both the location and eventually the function of the stress proteins.