Properties and purification of a glucose-regulated protein from chick embryo fibroblasts

141

Biochimica et Biophysica Acta. 576 (1979) 141--150 © Elsevier/North-Holland Biomedical Press

BBA 38048

PROPERTIES AND PURIFICATION OF A GLUCOSE-REGULATED PROTEIN FROM CHICK EMBRYO FIBROBLASTS

R O B E R T P.C. SItlU * and IRA H. PASTAN **

Laboratory o f Molecular Biology, National Cancer Institute. National Institutes o f Health, Bethesda, MD 20014 (U.S.A.) (Received May 9th, 1978)

Key words: Glucose; T~nsformation: Immunofluorescence: (Fibroblast. Rous sarcoma virus)

Summary A glucose-regulated protein of molecular weight 78 000 (GRP-78) had been purified from a membrane fraction isolated from viral transformed chick e m b r y o fibroblasts. Purification was achieved by extraction of the membrane fraction with Triton X-100, and chromatography on diethylaminoethylcellulose and hydroxyapatite. The purified protein exhibited one single spot on two-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has a pI of a b o u t 5.3. A monospecific antiserum to GRP-78 was generated in a goat. Immunofluorescence studies using affinity purified antibodies to GRP-78 revealed that this protein was not exposed on the cell surface but was localized in a granular vesicular network inside the cell that resembles the distribution of endoplasmic reticulum. The availability of purified GRP-78 and a specific antiserum to it should prove useful in elucidating the role of this protein in glucose metabolism and its relationship to malignant transformation. Introduction The synthesis of two membrane-associated proteins with apparent molecular weights of 95 000 and 78 000 is ~timulated in cultured chick e m b r y o fibroblasts and Balb 3T3 cells when they are deprived of glucose [1,2]. Further, the increased content of these two proteins in transformed cells, previously Abbreviat/ons: GRP, ~Juco~e-re~ul~ted proteLn; RSV-SR. Schmidt.Ruppm ~ n o f Rou~ ~ c o m a v i ~ SDS, s ~ i u m d ~ e c y l ~ a ~ . " P~nt ~d~; ~p~ment of Phydololy. Unive~ty of M ~ t o b ~ . Faculty of Medicine. Winnipeg. M ~ l t o b s . R3E OW3, C ~ a . " " T o whom c o ~ o n d e n c e ~ ~ add~d.

142 thought to be transformation specific [3,4], is principally due to the rapid depletion of glucose from the growth medium in which the transformed cells are grown [ 1]. The synthesis of these t w o proteins is suppressed in transformed cells by glucose and stimulated in normal, untransformed cells by the absence of glucose. We have designated these t w o proteins as glucose-regulated proteins (GRP); to date no biological role for these proteins has been identified. Since these proteins are present in substantial quantities in glucose-starved transformed cells, we feel they probably have an important role in these cells. With this idea in mind, we decided to isolate these proteins and prepare specific antibodies to them in order to help elucidate their biological functions. Here we report on the purification and characterization of one of these proteins that has a molecular weight of 78 000 (GRP-78). Materials and Methods Cell culture. Chick e m b r y o fibroblasts were cultivated, propagated and infected with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) as described elsewhere [ 5]. Purification o f GRP-78 Extraction. 30 roller bottles of fibroblast infected with RSV-SR virus were grown in glucose-free medium for 24 h before harvesting. All steps were carried o u t at 4°C. Each roller bottle of cells was rinsed twice with 50 ml of ice-cold Dulbecco's phosphate buffer saline containing 2 mM phenylmethylsulfonyl fluoride. Cells from 30 bottles were scraped into 500 ml of the same solution and homogenized in a tight-fitting Dounce homogenizer. The homogenate (878 mg protein) was centrifuged at 15 000 × g for 20 min. The pellet {250 mg protein) was resuspended in 300 ml phosphate-buffered saline/phenylmethylsulfonyl fluoride. Triton X-100 was added to a final concentration of 0.5% (v/v). After stirring for 10 min the suspension was centrifuged at 100 000 × g for 1 h. The supernatant solution contained 154 mg protein. Chromatography on concanavalin A-Sepharose. The detergent extract of membranes was allowed to run through a 10 ml column of concanavalin A-Sepharose (Pharmacia) previously equilibrated with phosphate-buffered saline (flow rate, 40 ml/h). Chromatography on DEAE-cellulose. The protein fractions that were not adsorbed by concanavalin A-Sepharose were dialyzed against three changes of 0.01 M NH4HCO3 (pH 7.8). The dialyzed solution was applied to a 15 ml column of DEAE-cellulose previously equilibrated with the above buffer (flow rate, 40 ml/h; 3.5-ml fractions). The column was washed with 100 ml 0.1 M NH4HCO3 {pH 7.8}. A linear gradient of 150 ml 0.1 M NH4HCO3 and 150 ml 0.4 M NH~HCO~ was applied. The column was finally eluted with 1 M NH~HCO3 buffer. For this and all subsequent chromatographic elutions, protein absorbance (A280nm) was monitored by a LKB Uvicord and ionic strength b y a conductivity meter. Chromatography on hj~droxyapatite. Fractions that contained GRP-78 after DEAE~ellulose chromatography were pooled {8.4mg protein), dialyzed against three changes of 5 mM potassium phosphate buffer (pH 7.0) and

143 applied on a 2 ml column of hydroxyapatite previously equilibrated with the same phosphate buffer (flow rate, 7 ml/h; 3-ml fractions). The column was then washed with 0.1 M phosphate buffer until the absorbance of the eluate approached zero. A gradient of 50 ml 0.1 M phosphate and 50 ml 0.4 M phosphate buffer was then applied.

Protein determination Protein was determined by the method of Lowry et al. [6] using bovine serum albumin as standard. Polyacry lamide gel electrophoresis One dimension. Throughout the entire purification, GRP-78 was monitored by SDS-polyacrylamide gel (in 7.5% gels, w/v) electrophoresis. Slab-gel electrophoresis was carried out using the procedures of Laemmli [7]. Two dimensions. Isoelectric focusing in the first dimension was performed in the presence of 8 M urea and ampholines (LKB), pH 3.5--10, as described by O'Farrell [8] and by Cabral and Schatz [9]. SDS-polyacrylamide gel electrophoresis in the second dimension was carried out on a slab with stacking, as described by Cabral and Schatz [9]. Radiolabeled proteins on the gel were detected by fluorography [ 10]. Generation o f antiserum to GRP.78 200 tzg purified GRP-78 was emulsified in complete Freund's adjuvant and injected intradermally at multiple sites in a goat; additional injections of 200 tzg GRP-78 in incomplete Freund's adjuvant were administered at multiple sites 3 and 6 weeks after the initial injection. The animal was bled 10 days after the third injection. Immunoprecipitation o f [3SS] methionine-labeled GRP- 78 We previously demonstrated that the goat antiserum is monospecific by an immunological gel localization technique using peroxidase-conjugated immunoglobulins [1]. To substantiate the specificity of the antibody a 150 mm dish of fibroblasts transformed by RSV was incubated at 37°C for 2 h with 8 ml Earle's balanced salt solution containing 10% (v/v) growth medium and 50 tzCi/ ml [3SS]methionine. After washing with Tris-buffered saline, cells were solubilized in 1% NP40, sonicated for 20 s and centrifuged for 100 000 × g for 1 h. A 25 ~zl aliquot of the supernatant solution was incubated with 5/A goat antiserum (heat-inactivated at 56°C for 30 min) in a final volume of 100 ~zl Tris-buffered saline, 0.5% NP40 2 mM phenylmethylsulfonyl fluoride. After 1.5 h at 4°C, 50 tzl 10% (w/v) suspension of Staphylococcusaureus (strain Cowan I), prepared as described by Kessler [11], were added. After another 30 min at 40C, the adsorbent was centrifuged and washed three times with Tris-buffered saline (pH 7.4), 2.5 M KC1, 0.5% NP40, 2 mM phenylmethylsulfonylfluoride. The immunoprecipitate was resuspended in 50 ~l 20 mM Tris-HC1 (pH 7.4), 8 M urea, 5% Triton X-100, 1% fl-mercaptoethanol, 2 mM EDTA. The bacterial adsorbent was removed by centrifugation. The immunoprecipitated, radiolabeled protein was analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis and visualized by fluorography as described earlier.

144

Localization of GRP-78 by indirect immunofluorescence using affinity purified antibodies Purification of antibodies to GRP-78 by affinity chromatography. 4 mg purified GRP-78 was coupled to 1 g CNBr-activated Sepharose 4B (Pharmacia}. More than 80% of the protein was coupled. After extensive washing, the coupled gel was diluted with non-activated Sepharose 4B to a final volume of 10 ml. A 7-globulin fraction was prepared from 60 ml goat anti-GRP-78 serum by precipitation using 35% saturated {NH4)2SO4. Washed three times with 35% saturated (NH4)2SO4 solution, the precipitates were redissolved in 50 ml phosphate-buffered saline and dialyzed against this buffer. This solution was then applied to the affinity column at 4°C (flow rate, 10 ml/h). The column was washed successively with phosphate-buffered saline, 0.1 M sodium borate (pH 8.2)/0.5 M NaC1 and 0.1 M sodium acetate (pH 4.9)/0.5 M NaC1 until the eluate was free of protein. Washing with the above two buffers was repeated. The column was finally eluted with 0.2 M acetic acid (pH 2.5)/0.5 M NaC1. This fraction contained highly purified antibodies to GRP-78 and was immediately dialyzed against phosphate-buffered saline. The initial washings with sodium acetate buffer resulted in the elution of some antibodies to GRP-78, but this fraction contained large amounts of non-specific immunoglobulins and therefore was not used. From 2.5 g protein applied to the affinity column, 6.8 mg of affinity purified antibody were obtained {0.25% recovery).

Immunoflourescence localization of GRP-78 Surface localization. SR-transformed fibroblasts still attached to substratum were f i x e d for 10 rain with 2% paraformaldehyde in 2% (w/v) sucrose. After being washed with phosphate-buffered saline, the cells were incubated for 15 min with 20 t~g/ml affinity purified antibody or 20 pg/ml non-specific immunoglobulins from a non-immune serum. After washing, cells were incubated with rhodamine-conjugated IgG (rabbit anti-goat, Cappel Lab), diluted 1 : 20 with Dulbecco's phosphate-buffered saline. Cells prepared in this manner were m o u n t e d in.glycerol buffer and examined using a Zeiss microscope equipped with epifluorescence optics. Exposure times were the same for all the photographs shown in Fig. 5. Intracellular localization. Monolayer cells still attached to the substratum were treated as described above except that the cells were permeabilized by t r e a t m e n t with 80% acetone in H20 for 10 min after the paraformaldehyde step. Results

Extraction o f GRP-78 A preliminary experiment was carried out to determine the subcellular localization of GRP-78 and the conditions required to extract it. Intact SRfibroblasts were treated by various reagents and the total cell extracts were centrifuged for 1 h at 100 000 ×g. The high-speed supernatant solution and pellets were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fig. 1A shows that only the nonAonic detergent Triton X-100 was

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Fig. 1. (A) E f f e c t s of v a r i o u s a g e n t s in the e x t r a c t i o n of G R P - 7 8 . M o n o l a y e r f i b r o b l a s t s t r a n s f o r m e d b y R S V - S R a n d s t a r v e d for glucose f o r 24 h w e r e w a s h e d t w i c e with D u l b e c c o ' s p h o s p h a t e - b u f f e r e d saline. One o f t h e f o l l o w i n g s o l u t i o n s was a d d e d t o e a c h dish: a, f, p h o s p h a t e - b u f f e r e d saline; b, g, p h o s p h a t e b u f f e r e d saline w i t h CaCI 2 (0.9 raM) a n d MgCI 2 (0.5 raM); c, h, p h o s p h a t e - b u f f e r e d s a l i n e / 0 . 0 1 % ( v / v ) T r i t o n X - 1 0 0 ; d, i, p h o s p h a t e - b u f f e r e d / 0 . 1 % T r i t o n X - 1 0 0 ; e, j, p h o s p h a t e - b u f f e r e d saline 2 M u r e a . All s o l u t i o n s c o n t a i n e d 2 m M p h e n y l m e t h y l s u l f o n y l fluoride. A f t e r 15 rain at r o o m t e m p e r a t u r e , cells w e r e s c r a p e d off t h e dish, h o m o g e n i z e d in a D o u n c e h o m o g e n i z e r a n d t h e h o m o g e n a t e c e n t r i f u g e d f o r 1 h at 1 0 0 0 0 0 X g at 4°C. T h e pellet was dissolved in 3% SDS; t h e s u p e r n a t a n t s o l u t i o n was d i a l y z e d e x t e n s i v e l y against 10 m M N H 4 H C O 3 , l y o p h i l i z e d a n d t h e c o n t e n t s dissolved in 3% SDS. P r o t e i n s w e r e a n a l y z e d b y S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . 25 #g p r o t e i n w e r e a p p l i e d to e a c h lane. a--e are 1 0 0 0 0 0 × g s u p e r n a t a n t s o l u t i o n s ; f--j are 1 0 0 0 0 0 × g pellets. L a n e k c o n t a i n e d p r o t e i n s t a n d a r d s : ( f r o m t o p ) m y o s i n ( 2 0 0 0 0 0 ) , p h o s p h o r y l a s e ( 9 4 0 0 0 ) , b o v i n e s e r u m a l b u m i n (69 0 0 0 ) a n d o v a l b u m i n (43 0 0 0 ) . T o p a r r o w i n d i c a t e s G R P - 9 5 a n d b o t t o m a r r o w i n d i c a t e d G R P - 7 8 . (B) S u b c e l l u l a r d i s t r i b u t i o n o f G R P - 7 8 . M o n o l a y e r f i b r o b l a s t s t r a n s f o r m e d b y R S V - S R p r e p a r e d as d e s c r i b e d in legends to A w e r e w a s h e d t w i c e w i t h D u l b e c c o ' s p h o s p h a t e - b u f f e r e d saline. Cells w e r e s c r a p e d off t h e dish in D u l b e c c o ' s p h o s p h a t e - b u f f e r e d saline, h o m o g e n i z e d a n d t h e h o m o g e n a t e c e n t r i f u g e d a t 10 0 0 0 X g for 15 m i n at 4°C. T h e p e l l e t was r e s u s p e n d e d in D u l b e c c o ' s p h o s p h a t e - b u f f e r e d saline a n d T r i t o n X - 1 0 0 was a d d e d to a final c o n c e n t r a t i o n o f 0.1% (v/v). A f t e r 5 rain stirring, t h e m i x t u r e was c e n t r i f u g e d at 1 0 0 0 0 0 × g for 1 h to o b t a i n a supern a t a n t s o l u t i o n a n d a pellet. With the e x c e p t i o n o f t h e final 1 0 0 0 0 0 X g pellet, all m a t e r i a l s w e r e d i a l y z e d against 10 m M N H 4 H C O 3 a n d l y o p h i l i z e d . E a c h f r a c t i o n was dissolved in a s o l u t i o n o f 3% SDS a n d t h e p r o t e i n s a n a l y z e d b y SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . 20 g g p r o t e i n was a p p l i e d t o e a c h lane. L a n e a, p r o t e i n s t a n d a r d s as in A; b, cell h o m o g e n a t e ; c, 10 0 0 0 × g s u p e r n a t a n t ; d, 10 0 0 0 × g pellet; e, 1 0 0 0 0 0 × g s u p e r n a t a n t f r a c t i o n o f T r i t o n X - 1 0 0 e x t r a c t f r o m 10 0 0 0 X g pellet; f, 100 0 0 0 X g pellet o f T r i t o n X - 1 0 0 e x t r a c t f r o m 10 0 0 0 × g pellet.

effective in the solubilization of GRP-78. When a homogenate of cells (in phosphate-buffered saline) was subjected to successive steps of centrifugation, almost all of the GRP-78 were found to be associated with the total membrane fraction; that is, the 15 000 × g pellet (Fig. 1B). When this 15 000 × g pellet was extracted with 0.1% Triton X-100 and the extract centrifuged at 100 000 × g, GRP-78 was found in the supernatant solution. Therefore, in the following

146 purification scheme, GRP-78 was initially extracted by 0.5% Triton from the 15 000 × g pellet of fibroblast homogenate. The resulting high-speed supernatant solution (100 000 × g for 60 min) was used as the starting material.

Chromatography on DEAE-cellulose The Triton extract that contained GRP-78 was first allowed to pass through a column of concanavalin A-Sepharose to remove glycoproteins and other carbohydrate-containing materials. (GRP-78 is not a glycoprotein since radioactive glucosamine, mannose and fucose are not incorporated into it (unpublished data)). This step, however, did not lead to significant purification of GRP-78. Fig. 2 shows the elution profile of GRP-78 and other proteins on the DEAEcellulose column. We consistently observed that GRP-78 eluted as a very broad peak that started at a b o u t 0.15 M NH4HCO3 and ended at a b o u t 0.3 M. Fractions that contained the majority of GRP-78 and the least a m o u n t of contaminants (fractions 60--85) were pooled and dialyzed. The DEAE-cellulose step also removed most of the Triton X-100; apparently its presence was not required to maintain GRP-78 in its soluble form. Chromatography on hydroxyapatite Fig. 3 shows the elution profile of GRP-78. It was eluted by 0.15--0.25 M potassium phosphate. Washing the column with 0.1 M salt prior to gradient elution removed practically all other proteins {fractions 1--3). Fractions 32--39 were pooled, dialyzed against 10 mM sodium phosphate buffer, and the final p r o d u c t was designated as purified GRP-78. From a total of 878 mg of cellular proteins, 3.3 mg of purified GRP-78 were obtained. Properties o f GRP- 78 Purified GRP-78, when analyzed on two-dimensional polacrylamide gel electrophoresis, exhibits one single spot with a pI of 5.3 (Fig. 4A). When puri-

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F ~ . 4. ( A ) T w ~ i m ~ o ~ p o l y ~ e ~1 e ~ p h o ~ of p ~ G~-7~. ~ ~1 (by Coom~e B ~ t Blue) ~ ~ o w n . (B) T ~ ~ o m ~ ~ i y ~ l ~ e ~! e ~ c ~ o ~ ot 35Sladled ceUu~ pro~ of flbrob~ ~ f o ~ by RSV~R. Auto,diem ~ ~own. A~w ~dlcs~ G R P - 7 8 . (C ~ d D) I m m ~ o p ~ p l ~ U o n of ~dio~bled GRP-78 by goat ~ m . A u ~ o ~ s m ~ o w n . (C) l m m ~ o p ~ c i p l ~ o n u~ aon-~mune soar ~m. (D) l m m u n o p ~ l D l ~ o n u~ soar ~UGRP-78 ~m; ~w radicals GRP-78.

148

fled GRP-78 was chroma~ographed on a column of Sepharose 6B that had been calibrated with protein standards, it eluted at a position that corresponded to a molecular weight of approx. 400 000. This suggests that GRP-78 may exist as a polymer in its native form. An antiserum against GRP-78 was obtained in a goat. To demonstrate that this antiserum is monospecific, we labeled transformed fibroblasts.with [3sS]methionine and used our antiserum to immunoprecipitate radiolabeled GRP78. Fig. 4B illustrates the profile of total labeled proteins of transformed fibroblasts on two-dimensional SDS gel electrophoresis; the arrow indicates GRP-78. When this cell extract was exposed to goat antiserum, two major spots were immunoprecipitated (Fig. 4D). One is GRP-78 (arrow); the other protein with a higher molecular weight was due to non-specific precipitation since it was also brought down by non-immune goat serum {Fig. 4C). As the function of GRP-78 is not known, information on its subcellular localization could give a hint about its functional role. Using affinity purified antibodies to GRP-78, we failed to demonstrate~its presence on the cell surface by indirect immunofluorescence (Figs. 5A and 5B). However, when the cells were permeabilized with acetone treatment, strong intracellular fluorescence was observed (Fig. 5D). The localization appeared as a granular, vesicular network in the cytoplasm. On higher magnification the fluorescence was not observed over the nucleus, mitochondria or lysosmal structures (identified by simultaneous phase microscopy). Preliminary studies indicate that it may be localized in a membranous network that resembles endoplasmic reticulum.

Fig. 5. L o c a l i z a t i o n o f G,RP-78 b y indirect i m m u n o f l u o r e s c e n c e using affinity purified a n t i b o d i e s ( A P A b ) . A a n d B, att.e~npt a t surface localization~ A , n o n - i m m u n e i m m u n o g l o b u l i n s ~ B~ affinity purified antib o d y . C and D, intracellular l o c a H z a t i o n ~ C, n o n - i m m u n e i m m u n o g l o b u l i n s : D, affinity purified a n t i b o d y .

149 More precise localization must await the results of ultrastructural antibody studies now in progress.

Discussion The protein GRP-78 that is in large amounts in glucose-starved cells and in particularly large amounts in glucose-starved transformed cells has been purified to near homogeneity by combining extraction with the non-ionic detergent, Triton X-100, and successive chromatography on ion~xchange resins. An antiserum to this protein was also generated. Immunofluorescence studies using antibodies to GRP-78 revealed that this protein is not exposed on the cell surface. This probably accounts for our unpublished observation that this protein cannot be iodinated by the lactoperoxidase procedure. GRP-78 is localized, however, in a membranous network inside the cell that resembles the distribution of endoplasmic reticulum; however, the present t ~ h n i q u e c a n n o t rule out the possibility that this protein could be on the inner surface of the plasma membranes. At present, the functional role of GPR-78 remains obscure. Several investigators [12,2,1] suggested that it may have a role in glucose transport b ~ a u s e it was observed to accumulate in transformed cells and glucose-s -tarred cells. The content of another protein of molecular weight 95 000 (GRP-95) is "also increased in a manner similar to that of GRP-78. The increased content of GRP-78 and GRP-95 is always associat~.~i with incre&~ed rates of glucose transport as occurs after glucose starvation and transformation [ 1 3 - 1 5 ] . Recently in our laboratory, Pouyssegur and Yamada (unpublished data) have isolated GRP-95 and obtained an antiserum to it. Using immunofluorescence procedures, they found that GRP-95 was localized on the cell surface and can be iodinated by lactoperoxidase~:atalyz~t reaction. Evidence to suggest that GRP-95, GRP-78, or both may be involved in glucose transport came from a recent study of Shanahan and Czech [ 16,17]. These inw~stigators were able to extract two proteins with molecular weights 94 000 and 78 000 from adipocytes. When these two prou~ins were reconstituted into phospholipid vesicles, glucose transport activity was observed. In light of these studies, it is quite possible that GRP-78 and GRP-95 are proteins of the glucose-transport complex. Acknowledgments We thank Drs. Gilbert Jay and Mark Willingham for their helpful suggestions and Miss E. Lovelace for propagating the cells used in this study. We also thank Drs. Shanahan and Czech for sending us their preprint. R.P.C.S. is a Centennial Fellow, Medical Research Council of Canada.

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