Prosomes and Heat Shock Complexes in Drosophila ... - Europe PMC

MOLECULAR AND CELLULAR BIOLOGY, June 1989. p. 2672-2681 0270-7306/89/062672-10$02.00/0 Copyright ©D 1989, American Society for Microbiology

Vol. 9, No. 6

Prosomes and Heat Shock Complexes in Drosophila melanogaster Cells SA,1 EMMANUELLE ROLLET,2 MARIA-FATIMA GROSSI DE SA,1 ROBERT M. TANGUAY,3 MARTIN BEST-BELPOMME,2 AND KLAUS SCHERRER1* Institiut Jacques Monod, 2 Place Jiussieiu, 75251 Patris Cedex 05, France1; UA Centre National de la Recherche Scientifique, 75251 Paris Cedex 05, France2; and Ontogenese et Gnentique MoleMcilaire, Centre Hospitalier de l'Universite de Laval, Ste Foy, Quebec GI V 4G2, Canada3 CEZAR MARTINS DE

Received 9 August 1988/Accepted 1 February 1989

Prosomes and heat shock protein (HSP) complexes isolated from the cytoplasm of Drosophila cells in culture were biochemically and immunologically characterized. The two complexes were found to separate on sucrose gradients, allowing the analysis of their protein constituents by two-dimensional polyacrylamide gel electrophoresis and by reaction with anti-HSP sera and prosome-specific monoclonal antibodies. All of the prosomal proteins were found to be clearly distinct from the HSP; none of the prosomal proteins was synthesized de novo in heat shock. However, an antiprosome (anti-p27K) monoclonal antibody (mouse anti-duck) recognizing the Drosophila p29K prosomal protein allowed immunoprecipitation from a heat-shocked postmitochondrial supernatant of the crude HSP complex, including the low- and the high-molecular-weight components, in particular the 70 x 103-molecular weight HSP. The highly purified small 16S HSP complex still contained this preexistent p29K prosomal protein, which thus also seems to be a metabolically stable constituent of the HSP complex. The significance of this structural and possibly functional relationship between prosomes and HSP, involving the highly ubiquitous and evolutionarily conserved prosomal protein p27/29K, remains to be elucidated.

Having noticed for some time a similarity between this protein pattern and that of a subgroup of cytoplasmic messenger ribonucleoproteins (mRNPs) later identified as belonging to the prosomes (e.g., Fig. 4A' and B' in reference 51), we drew attention to a possible interrelation of the two types of cytoplasmic small RNP complexes, the HSP and the prosomes (42). Prosomes are a novel kind of ubiquitous cytoplasmic RNP and subcomplexes of free mRNP. These particles of characteristic morphology were first observed by electron microscopy in RNP fractions of HeLa cells (45) and were later identified as being a biochemical entity of their own (42). Prosomes are composed of 85% proteins and 15% RNA (pRNA). They represent a morphologically and biophysically uniform class of 12-nm-wide raspberry-shaped particles which vary, however, in individual protein and RNA composition, function of the cell type (35), and the mRNA population with which they are associated (32). A minimum of 25 prosomal proteins and about as many different pRNAs were observed in various cells and species. Use of prosomespecific monoclonal antibodies (MAbs) revealed that prosomal antigens can also be observed in the nucleus (21), where they associate with the transcribed chromosomes and the nuclear matrix (35). Furthermore, the prosomes, and thus the mRNP, were found to be located on the intermediate filaments of the cytoskeleton (22). Some of the prosomal proteins are phosphorylated (52), ADP ribosylated (47), or glycosylated (48), and some have a protease activity (46; Nothwang, Buri, and Scherrer, unpublished observations). However, contrary to a view recently expressed by Falkenburg et al. (19) and Arrigo et al. (5), the characteristics and functions of the prosomes cannot simply be accounted for by those of a well-known protease (14) devoid of RNA. They are a multicomponent RNP strongly associated with mRNA (1, 32) and inhibit protein synthesis in vitro (1). They were found in all cells tested but exist in

The heat shock response of most eucaryotic cells involves a strong increase in the synthesis of a small set of evolutionarily conserved proteins known as the heat shock proteins (HSPs). A similar response is induced by a wide variety of other stress agents (for reviews, see references 6, 31, and 41). In eucaryotic cells, HSPs have also been shown to be induced by ethanol (29), by arsenite (53), in recovery from anoxia (38, 39), by cadmium (12), and by hydrogen peroxide (13). The predominant inducible HSPs can generally be classified into three major groups based on their molecular sizes of 80 to 90, 65 to 75, and 20 to 30 kilodaltons (kDa). The last group (referred to as the low-molecular-weight HSPs) is especially well characterized in Drosoplila cells. It has been postulated that these proteins play an important role during embryonic development (7), hormonal induction in Drosophilia melanogaster (23), and normal cell growth (56) and that HSPs have a protective effect (50). However, their precise cellular function remains obscure (41, 55; see also Discussion). The small HSPs were shown to exist as a complex in the cytoplasm, forming a characteristic band in density gradients including protein and RNA (2-4). Some of the HSPs were thought to be primarily associated with the nucleus during the heat shock (24). More recent data of Bonner and collaborators (27) indicate, however, that the HSPs are associated with the intermediate filaments of the cytoplasm and that upon heat shock, these cytoskeletal elements collapse onto the nuclear membrane. Therefore, it seems that the small HSPs exist essentially in the cytoplasm and not in the nucleus, as assumed previously on the basis of the fact that after heavy stress, they co-isolate with the nuclei. The most heterogeneous group of HSPs is constituted by small proteins in the 20- to 30-kDa range which form a characteristic pattern in mono- and bidimensional gel electrophoresis. *

Corresponding author. 2672

PROSOMES AND THE SMALL HEAT SHOCK PROTEINS

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various combinations of RNA and protein and show a most interesting differential pattern of cytolocation in oogenesis (20), development (35), and differentiation (21). As for the alleged identity of the HSPs and prosomal proteins (3, 43) dealt with in this paper, the significance of the protease activity of prosomes, which recalls that of factors as various as mammalian growth factors (34) and the Gro E factor of Escherichia coli (8), for example, will need extensive and careful investigation (in progress). Extensive biochemical and cytological correlations thus seemed likely to exist between prosomes and HSPs, particularly in view of the selective protein patterns. Prompted by our suggestion (42), two groups (3, 43) reported evidence that prosomal proteins and HSPs which copurify are related. Comparing the cytoplasmic prosomal 23-kDa protein to the nuclear HSP 23, Schuldt and Kloetzel (43) showed that not only the immunological determinants but also the peptide patterns generated by the V8 protease are similar. In view of all these data, we undertook to investigate in detail this possible relationship between prosomes and the HSP complex. It is shown here that the two types of complexes can be separated and purified from Drosophila lysates and that none of the prosomal proteins is a genuine HSP or actively synthesized under heat shock conditions. However, the protein p29K seems to be a stable and basic constituent of the heat shock complex, as well as of prosomes, closely associated with the HSP but not synthesized de novo under heat shock conditions.

MATERIALS AND METHODS

Drosophila cell culture. The 89K cellular clone used in these studies is derived from the D. melaniogaster KC line (17). The cells were seeded into 25-cm2 flasks and grown at 23° C in D22 medium containing 5% decomplemented fetal calf serum (18). Heat shock treatment and cell labeling. Cells were heat shocked following 2 days of subculture; they were incubated at 37° C for 2 h in D50 medium (D22 minus yeast extract, lactalbumin, and methionine) in the presence of 100 p.Ci of [35S]methionine (SJ204; Amersham Corp.) per ml. For pulsechase studies, cells were labeled as described above and then chased for 6 h at 23° C in cold medium. Labeling was stopped by putting the flasks on ice. Cells were then detached gently with a rubber policeman and centrifuged for 10 min at 800 x g, and the cellular pellet was washed in isotonic S22 buffer (10 mM Tris [pH 7.4], 110 mM NaCI, 80 mM

KCI).

Cell fractionation. Cell pellets were suspended in 8 vol(10 mM Tris-ethanolamine [pH 7.4], 10 mM NaCl, 1 mM MgCl,, 5 mM mercaptoethanol) containing 0.4% Nonidet P-6 and 1 mM phenylmethylsulfonyl fluoride. After 10 min on ice, the cells were homogenized in a Dounce homogenizer; lysis was checked by phase-contrast microscopy. Nuclear pellets were obtained by 10 min of centrifugation at 1,000 x g. The postmitochondrial supernatant (20 min, 10,000 x g) was centrifuged for 90 min at 150,000 x g on a 20% sucrose cushion to pellet polyribosomes. The last supernatant was recentrifuged for 18 h at 150,000 x g to pellet the free cytoplasmic RNP complexes. Preparation of an antibody against HSP 23. The synthetic umes of TENM2

oligopeptide (Val-Lys-Glu-Asn-Pro-Lys-Glu-Val-Glu-GlnAsp) encoding the amino acid residues 170 to 181 of D.

melanogaster HSP 23 was obtained from the Institut Ar-

mand Frappier (Division of Chemical Products, Montreal, Quebec, Canada). The carboxy-terminal region of HSP 23

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was chosen on the basis of its lack of homology with the other three small HSPs (44). The peptide was coupled to keyhole limpet hemocyanin using sulfosuccinimidyl 4-p-maleimidophenylbutyrate (Pierce Chemical Co.). Rabbits were immunized with 250 Rg of coupled peptide and incomplete Freund adjuvant. The specificity of the antibody was tested by immunoblotting (see Results). Fractionation of free RNP complexes. The pellets were suspended in 0.5 to 1.0 ml of low-ionic-strength TEK buffer (10 mM TEA [pH7.4], 50 mM KCI, 5 mM mercaptoethanol) and sedimented on a 5 to 27.5% (wt/wt) sucrose gradient in high-ionic-strength TEK buffer (10 mM TEA [pH 7.4], 500 mM KCI, 5 mM mercaptoethanol) for 18 h at 150,000 x g. Fractions (0.4 ml) were collected by using an Isco gradient fractionator with a Noll-type continuous-flow cell, and the A,54 was recorded. CsCI gradient centrifugation. The interesting fractions from the sedimentation gradient were mixed with the bottom layer of a CsCl gradient. After centrifugation (Beckman rotor SW60; 40,000 rpm, 20° C, 20 h), the gradient was fractionated, and the radioactivity present in each fraction was counted. Gel electrophoresis of proteins. Electrophoresis in one dimension was performed by the method of Laemmli (25). Nonequilibrium electrofocusing and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) were performed as described by O'Farrell et al. (33). The mixture of ampholines in the first dimension was 0.4% ampholines (pH range, 5 to 7: 0.2% ampholines from LKB Instruments, Inc.. plus 0.2% from Pharmacia) and 1.6% ampholines (pH range, 3 to 10; 0.8% LKB ampholines plus 0.8% from Pharmacia). Electrofocusing was performed at 800 V for 3 h. Molecular weight markers used in the second dimension were phosphorylase b (92,000), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,000), and lactalbumin (14,000). Peptide mapping by the V8 protease. Digestion was by the method of Cleveland et al. (10). Interesting spots were cut from dried two-dimensional gels. After equilibration, they were loaded on an 18% acrylamide gel in the presence of 0.3 ,g of V8 protease. After the proteins migrated into the stacking gel, the current was stopped for 30 min to allow the reaction to occur. Then migration was carried out, and the gel was dried and exposed for autoradiography. Electrophoretic blotting and immunological detection of proteins. After electrophoresis, proteins were transferred onto nitrocellulose sheets (0.45-,um pore size; Schleicher & Schuell, Inc.) following the method of Towbin et al. (49). The paper was saturated overnight at 4° C in phosphatebuffered saline (PBS) containing 5% nonfat milk to eliminate nonspecific reactions. It was then incubated with the purified anti-p27K (21) MAb (1/500 dilution) or the polyclonal antibody alpha-23 (1/1,000 dilution) for 3 h at room temperature. After three washings in PBS, the filter was incubated with a peroxidase-labeled rabbit anti-mouse antibody diluted in PBS (1/1,000) and containing 10% normal rabbit serum. After this reaction, the filter was washed three times in PBS and developed with 4-chloro-1-naphthol and H,O,. Immunoprecipitation. The [35S]methionine-labeled postmitochondrial supernatant was incubated with mouse preimmune serum fixed on protein A-Sepharose in NET2 buffer (10 mM Tris [pH 7.4], 150 mM NaCI, 0.5% Nonidet P-40). After 20 min at room temperature, the supernatant was recovered and the procedure was repeated twice in order to eliminate nonspecific precipitation. The last supernatant was incubated with anti-p27K (21) MAb lB5 or polyclonal alpha-

2674

MARTINS DE SA ET AL.

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FIG. 1. Coimmunoprecipitation from a postmitochondrial lysate of small HSPs by the anti-p27K prosomal MAb and by the alpha-23 serum specific to the Drosoplila HSP 23. KC cells (clone 89K) were grown in monolayer culture and labeled at 37 or 23° C as described in Materials and Methods. (A) Fluorograms of immunoprecipitates. The postmitochondrial supernatant from heat-shocked cells prepared, and the specific proteins in the immunoprecipitates were analyzed by SDS-PAGE and fluorography. Lanes: 1 and 4, control reactions using nonimmune serum; 2 and 3, immunoprecipitates using the anti-p27K MAb (recognizing the p29K Drosopilai prosomal protein); and 5 and 6. anti-HSP alpha-23 serum. To destroy protein complexes. the samples analyzed in lanes 3 and 6 were adjusted to 0.5Q% SDS, heated to 600C for 5 min, and diluted five times prior to immunoprecipitation. (B) The postmitochondrial supernatants from normal and heat-shocked cells were fractionated by SDS-PAGE and blotted onto nitrocellulose paper. Laines: 1 and 2. fluorograms of the blot; 3 and 4. reactions with the anti-p27K MAb; 1 and 3, normal cells: 2 and 4. heat-shocked cells. (C) Lysates from normal aind heat-shocked cells were fractionated by SDS-PAGE. Lanes 1 and 2. Fluorograms of the gel. An identical gel was blotted onto nitrocellulose paper. Lanes 3 and 4. The reaction with the anti-hsp 23 (alpha 23 serum) polyclonal antibody. Lanes 1 and 3. Normal cells. Lanes 2 and 4. Heat-shocked cells. was

23 serum for 20 min at room temperature. Protein ASepharose was added, and the reaction was continued overnight at 4° C. The mixture was pelleted, and the pellet was washed five times with 1 ml of NET2 buffer containing 0.1%, SDS, suspended in 30 .1I of Laemmli loading buffer. and analyzed by SDS-PAGE. RESULTS Immunoprecipitation of the HSP complex by the anti-p27K prosomal MAb and the anti-HSP 23 serum. MAbs raised against duck prosomal proteins were produced and found to recognize highly conserved prosomal epitopes in several species, in particular, Drosophlila cells (22). These observations prompted us to use an anti-p27K MAb as a reagent in lysates of heat-shocked Drosophila cells in order to test for a possible relationship between prosomes and small HSPs (3, 42, 43). The anti-HSP 23 serum was produced against a synthetic peptide derived from a nonconserved domain of the HSP 23 gene product specific to this protein. Thus, it seems unlikely that the antibody alpha-23 might react with other HSPs. This antibody recognized HSP 23 exclusively and no other protein (Fig. 1A, lane 6; Fig. 1C). The prosomal p27K protein is a ubiquitous component of the cytoplasmic prosomes in avian and mammalian cells (32). During short periods of incubation and at normal temperature, as in this experiment. prosomal proteins are barely labeled by [35Slmethionine. Figure 1A shows immunoprecipitations using the antip27K MAb as well as the anti-Drosophila HSP 23 serum (alpha-23 serum). After electrophoresis, immunoprecipitates of 35 S-labeled postmitochondrial supernatants of heatshocked cells with the anti-p27K MAb (Fig. IA. lane 2)

showed prominent protein bands in molecular weights corresponding to those of all known major HSPs, HSP 27/26 and HSP 23/22. as well as the 70- and 83-kDa components (note that in this gel system, the small HSPs were not resolved). When the anti-HSP alpha-23 serum was used, proteins showing an identical pattern were evident (Fig. IA, lane 5). Controls using nonimmune serum (Fig. IA, lanes 1 and 4) show that these immunoprecipitations are specific, it should be noted that these two antibodies immunoprecipitate a substantial fraction but not all of the labeled proteins. To explain the fact that prosomal anti-p27K MAb (antiduck) and the anti-HSP 23 serum produced the same pattern of immunoprecipitated proteins, two hypotheses can be put forward. Either (i) prosomal protein p27K and HSP 23 fortuitously share an epitope common to all Drosoplhilai HSPs or (ii) prosomal p27K and HSP 23 are part of a highly heterogeneous (cytoskeletal) complex including, in stressed cells, all HSPs and many more labeled and unlabeled proteins. The latter explanation seems more likely since (i) SDS at a low concentration abolished precipitation of the HSP by the prosomal MAb (Fig. 1A, lanes 3 and 6), whereas although the four small HSPs were synthesized in the heatshocked cells (Fig. 1A and B, lanes 2). none of them was recognized by the prosomal antibody (see Fig. 4B, lane 4). The hypothesis that p27K and one or several of the small HSPs have an epitope in common could thus be ruled out. To analyze further the relationship between these proteins, a postmitochondrial supernatant was prepared from [35Slmethionine-labeled cells. either control cells or heatshocked cells: the proteins were fractionated by SDS-PAGE

and blotted onto nitrocellulose paper. Reaction with the anti-p27K MAb showed (Fig. lB, lanes 3 and 4) that in both

VOL. 9. 1989


preparations, this antibody, which is specific to a 27-kDa protein in mammals, recognized in Drosoplhilai cells a single protein with a molecular weight of 29,000 and none of the labeled HSPs. Indeed, autoradiography of the same blot (lanes 1 and 2) revealed that this 29-kDa protein was not preferentially labeled during heat shock. It is hence not an HSP in Drosoplhila cells in terms of molecular weight and level of synthesis under heat shock conditions. The p27K prosomal protein is a quite stable cytoplasmic prosomal component and thus not labeled in a short pulse (2 h) as done here. Therefore, it is undetectable in our immunoprecipitations (Fig. 1A, lanes 2 and 5), although present in both precipitates as an unlabeled component reacting with the anti-p27K MAb (data not shown). The fact that under stress conditions the p29K protein recognized by the antip27K MAb could not be detected by fluorography shows that it is not an HSP. These findings are consistent with the notion that some of the small HSPs are involved in the formation of a macromolecular complex which seems to include the p29K prosomal protein. This complex might thus be related to the prosomes. Separation of prosomes and the HSP complex. Postmitochondrial supernatants of normal and heat-shocked cells were fractionated by differential centrifugation in order to produce polyribosomes, pellets of postpolyribosomal particles, and the cytosol fraction. SDS-PAGE of these fractions (not shown) indicated that small HSPs cofractionate mainly with the postpolyribosomal pellet, which is referred to as the free mRNP fraction (16, 32, 42, 52). Prosomes have been successfully dissociated and purified from this free mRNP fraction by sucrose gradient sedimentation in high-ionicstrength buffers (32, 42). Under these conditions, the prosome sediments as a sharp peak with a sedimentation value of about 19S. The same protocol was used in an attempt to separate HSPs and prosomal components. Free mRNP fractions from [35S]methionine-labeled control and heat-shocked Dr-osophila cells were sedimented in sucrose gradients containing 0.5 M KCl. A systematic analysis by SDS-PAGE of each fraction of such a gradient is shown in Fig. 2. Drosoplhila KC cells (clone 89K) were grown in monolayer culture and labeled by 100 ,uCi of [35S]methionine for 18 h at 23° C (control cells) or for 2 h at 23° C, followed by 1 h of incubation at 37° C (heat-shocked cells). Cells were lysed, and free mRNP fractions were prepared as described in Materials and Methods. The mRNP pellets were dissolved in TEK buffer containing 0.5 M KCI, and the same quantity of material absorbing at 260 nm was sedimented through 5 to 21% (wt/wt) isokinetic sucrose gradients in the same buffer (Beckman SW41 rotor; 37,000 rpm, 17 h, 4° C). Fractions (0.4 ml) were collected from the bottom of the tube, and 60-,ul samples were trichloroacetic acid precipitated (10% final concentration [vol/vol], 30 min, on ice), collected by centrifugation, washed with acetone, and dissolved in SDS buffer prior to electrophoresis on 13%Y polyacrylamide slab gels. When the mRNPs of the normal and heat-shocked cells were compared (Fig. 2), no major differences were detected in the Coomassie blue-stained gels (Fig. 2A). but a striking change was visible in the fluorograms of the same gels (Fig. 2B). Typical prosomal proteins could be seen in the 19S region of both of the gels, whereas the HSPs could be observed in the extract of heat-shocked cells only. The labeled small HSPs sedimented in two main peaks in the 16S and 12S regions of the gradient with the heat-shocked RNPs: analysis under low-ionic-strength conditions gave the same results (not shown). A prominent protein of about 90 kDa

2675

sedimented with the 16S complex; however, since its distribution did not exactly coincide with that of the 16S complex, the question remains as to whether the 90-kDa protein belongs to the same entity as the 26/27- and the 22/23-kDa HSPs. This protein was not labeled to the same extent as the HSPs and did not immunoprecipitate with either the antip27K MAb or the alpha-23 anti-HSP serum (Fig. 1B). These results indicate that prosomes and the aggregates including the small HSPs are different entities, which can be separated in our experimental conditions. Further data supporting this suggestion are illustrated in Fig. 2C. Fractions of both gradients were electrophoresed, blotted onto nitrocellulose paper, and then reacted successively with the antip27K MAb and the alpha-23 serum. As expected, the alpha23 serum gave little or no reaction on the blots of extracts from control cells, whereas the anti-p27K MAb reacted with the p29K prosomal protein in the 19S region and with a band of low intensity in the 16S region (Fig. 2C, left panel). In the blot of heat-shocked cells (Fig. 2B, right panel), HSP 23 could be detected by the alpha-23 serum in the 16S and 12S regions. Again, the anti-p27K MAb reacted with a 29-kDa prosomal protein in the 19S and 16S regions and, interestingly, also with a protein at the top of the gradient which reacted strongly in comparison with the control blot. It should be noted that under none of the conditions tested thus far was a prosomal protein observed outside the 19S particle: the appearance of a protein as an unlabeled component of 4S implied the disassociation of preexisting prosomes in heat shock. In view of these results, the question arises as to whether the 12S and 16S HSP complexes are in dynamic relation to each other and, furthermore, whether the HSP complexes containing the preexisting p29K protein are precursors of the 19S prosome particles. To test the dynamics of formation of both types of HSP complexes (16S and 12S), cells were heat shocked at 37° C for 2 h in the presence of `'S]methionine and allowed to recover for 6 h in cold medium at 23° C. Fractions of mRNP were processed as described above, and fluorograms of the electrophoresis of control, heat-shocked, and chased cells were examined (Fig. 3). Under such heat shock conditions, prosome synthesis stopped and both the 16S and 12S regions containing small HSPs were strongly labeled (Fig. 3B). Fluorography of the chased radioactivity revealed that the 16S complex was more stable than the 12S complex: about 80% of the labeled small HSPs were found in the complex sedimenting in the 16S region (Fig. 3C). For this reason, the 16S complex was used for further investigation and will be referred to as the 16S HSP complex. The 19S prosomes did not show any label in these conditions; we thus conclude that there is no dynamic relationship between the HSP complexes and the prosomes. None of the prosomal proteins is a genuine HSP, but the highlv purified 16S HSP complex contain the prosomal protein p29K. Prosomes and the 16S HSP complex were further purified by CsCl density gradient centrifugation without fixation. Like the prosomes (42). the HSP complex resists high ionic strength (see Materials and Methods). Prosomes and the 16S HSP complex containing fractions from normal and heat-shocked cells purified on sucrose gradients (Fig. 2) were mixed with the bottom layer of the respective CsCl gradient and centrifuged. The density of prosomes was 1.3 g/ cm 3, and the density was about 1.33 g/cm3 for the 16S HSP complex. These values indicate that both complexes contain nucleic acids; indeed, RNA could be identified in both (3. 32; results not shown).

2676


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To characterize better the protein moiety of these complexes, two-dimensional gel electrophoresis of the pooled fractions of the density gradients was performed. Figure 4 shows a comparative analysis of the labeled proteins found in the fractions previously separated on sucrose gradients and repurified as described above. Sucrose gradient fractions containing either prosomes or the 16S HSP complex, respectively, from normal and heat-shocked cells were pooled and mixed with the bottom layer of a CsCl2 density gradient and centrifuged (Beckman SW60 rotor; 40,000 rpm, 1 h, 20° C). Fractions (0.2 ml) were collected, and their radioactivity was determined. The material in the main peak

of radioactivity was pooled and analyzed by electrophoresis as described in Materials and Methods. The fluorographs of the gels show that there were no differences in the protein patterns of purified prosomes from normal and heat-shocked cells (Fig. 4A and C). Neither the composition nor the labeling of prosomal proteins changed upon heat shock, compared with normal conditions. However, the protein pattern of the 16S HSP complex (Fig. 4B and D) was found to be quite different. Under normal conditions (23° C), the 16S complex contained a few detectable proteins, the major 90-kDa component discussed above and a few other spots in the 50-kDa range. At 37° C, the


VOL. 9, 1989

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FIG. 3. Stability under chase conditions of small HSPs in aggregates of various sizes. Pellets of mRNPs were dissolved in TEK buffer containing 0.5 M KCI and sedimented through a sucrose gradient, and fractions of the gradients were analyzed as described in the legend to Fig. 2. (A) Control. Cells were labeled at 23° C for 18 h. (B) Heat shock. Cells were labeled at 37° C for 2 h. (C) Chase. Cells were labeled at 37° C for 2 h, and the label was chased for 6 h at 23° C in cold medium. The fluorogram of the zone corresponding to molecular weights of 21.000 to 45,000 is shown.

pattern was greatly changed, and the four low-molecularweight HSPs were highly labeled. Note that none of these four proteins was present in heat-shocked prosomes; this is additional proof that prosomes and HSPs are different and also that the prosomes and the 16S HSP complex

can

separate successfully. Prosomal proteins might behave like normal proteins, i.e., their synthesis might not be increased at 37° C, but they could, on the other hand, totally or partially share the same amino acid sequence with HSPs. This was suggested by results reported by others (43). To test for this possibility, we did fingerprinting of the peptides created by V8 protease to compare the proteins most likely to be similar in the two complexes. Prosomal proteins migrating in two-dimensional electrophoresis to identical or closely similar positions were chosen from control cells and from the 16S HSP complex of heat-shocked cells (Fig. 4, arrows). HSP 23 (Fig. 5, lane b) was compared with the two prosomal proteins (outlined by arrows in Fig. 4) in the 23-kDa molecular size range (Fig. 5, lanes a and c). The patterns of peptide mapping seem clearly different (compare lane b with lanes a and c). The same results were obtained for proteins in the 26- to 27-kDa range. HSPs 27 and 26 were compared with a prosomal protein of 27 kDa; the peptide patterns were also different (compare the HSPs in lanes d and f with that in lane e). These results indicate that these prosomal proteins are not identical to HSPs synthesized under normal conditions. Finally, additional evidence was obtained that the prosomal Drosophila protein p29K possibly participates in the formation of the 16S HSP complex (Fig. 1 and Fig. 2C). To

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test this notion further, the 16S HSP complex, highly purified by CsCl gradient centrifugation, was fractionated by SDS-PAGE and blotted onto nitrocellulose paper and the blot was reacted with the anti-p27K MAb. The anti-p27K MAb stained a band of 29 kDa present in the purified 16S HSP complex (Fig. 6W, lanes 2 and 3) of normal (lane 3) and heat-shocked cells (lane 2). The presence of the prosomal protein p29K in the 16S HSP complex suggests that it is in physical association with the small HSP, forming a preexisting component of the HSP complex. Association to the nucleus by the prosomal p29K protein and the HSPs under conditions of heat shock. Based on the observations of Leicht et al. (27) and Collier and Schlesinger (11) reporting the collapse of the HSP onto the nucleus, the cytolocalization of the prosomal p29K protein compared with that of the HSPs was investigated. Cells were subjected to heat shock and labeled under the conditions given in the legend to Fig. 3 or during recovery after a total of 6 h at high temperature. The cells were fractionated into soluble fraction, cytoskeleton, chromatin, and nuclear matrix by the protocol of Penman (28) and analyzed by SDS-PAGE and immunoblot by using the anti-p27/29K prosomal MAb. At normal temperature the p29K protein is essentially cytoplasmic, while in heat shock it also apparently becomes associated to the nuclear matrix fraction (Fig. 7W; lane H in MAT). The same holds true for the low-molecular-weight as well as the high-molecular-weight HSPs (panel A, lane H in MAT) as reported previously by Leicht et al. (27). It is most interesting that the p29K antigen, associated to the nuclear matrix in stress conditions, was released upon recovery. The same holds true for the HSPs themselves. These data can be accounted for by two models. (i) The HSP complex, including the p29K prosomal antigen, becomes associated as a unit to the nuclear fraction upon collapse of the cytoskeleton in stress. (ii) The HSP and the p29K antigen are both attached to the cytoskeleton but as independent units and are shifted to the nuclear matrix owing to the collapse of their common support.

DISCUSSION One of the main new observations reported here concerns the fact that the prosomal protein p29K of D. inelanogaster (p27K in most vertebrates) is also a constituent of the HSP complex. Furthermore, it was found that the four small HSPs constitute a complex which is distinct and separable from the prosome. However, contrary to our suggestion (42) and to the conclusions of Arrigo et al. (3) and Schuldt and Kloetzel (43), it became obvious that none of the prosomal proteins was an HSP. In particular, the p29K protein was not labeled in heat shock. However, it behaved as though it were part of the HSP complex, in particular, like the HSP complex, it became part of the nuclear fraction during heat shock. Therefore, a structural and possibly functional interrelation of prosomes and the HSP complex may exist. In the work presented here, we were able to differentiate these two complexes by the following five criteria. (i) Their sedimentation constants were different, 19S for the prosomes and 12S and 16S for the HSP complexes. (ii) Their buoyant densities were different, 1.3 g/cm3 for the prosomes and 1.33 g/cm' for the HSP complex. (iii) The four lowmolecular-weight HSPs were present only in the 16S complex, whereas no newly labeled protein appeared in the prosomes after heat shock. (iv) V8 peptide mapping analysis shows a different pattern when individual HSPs and proso-

2678


MOL. CELL. BIOL. N E PH G E _.

B

A

3 1.

'

9

2 1 1 4 0

D

C

-

9 2 66 4 5-

3 12l_ 1 4

FIG. 4. Comparison by two-dimensional PAGE of the low-molecular-weight proteins found in the 16S HSP complex and the prosomes. Cells were labeled and processed as described in the text and the legend to Fig. 2. Sucrose gradient fractions containing either prosomes or the 16S HSP complex, respectively, from normal and heat-shocked cells were analyzed by electrophoresis as described in Materials and Methods. Fluorograms of the gels are shown. (A) Prosomes from control cells. (B) 16S zone of control cell extracts. (C) Prosomes from heat-shocked cells. (D) 16S HSP complex from heat-shocked cells. The arrows indicate the protein spots (in panel A, p22K, p23K, and p27K; in panel D, HSP 23 and HSP 27) cut out from the gel for subsequent peptide mapping (Fig. 5). NEPHGE, Nonequilibrium pH gel electrophoresis.

mal proteins of similar molecular weights and pIs are compared. (v) The 16S HSP complex contains the two HSPs that were shown to be phosphorylated upon heat shock (37), but they were undetectable in the prosomes isolated from in vivo a

0

b

c

d

e

f

31 2114-

9

a

2

FIG. 5. Comparative peptide mapping of the prosomal and small HSPs. Proteins identified in Fig. 4 (arrows) were cut out of the dried gel and subjected to partial proteolytic cleavage by the protease V8 and fractionated as described in Materials and Methods. Fluorograms of the gels are shown. Lanes: a, prosomal protein in p22K; b, HSP 23; c, prosomal protein p23K; d and f, HSP 27; e, prosomal protein p27K.

32P-labeled heat-shocked cells (data not shown). We can thus conclude that the prosomes and the HSP complex are two biochemically distinct and separable kinds of RNP complexes in Drosophila cells. Upon heat shock, the four small HSPs were labeled in two distinct regions of the gradient, 16S and 12S. Labeling during heat shock followed by a 6-h chase in non-radioactive medium showed a predominant labeling of the HSP in the 16S region. The meaning of this is not clear, but several hypotheses can be put forward. First, the HSP complex might be a dynamic system which is suddenly stopped in the course of the experiment. Keeping this in mind, one may suggest that the 12S complex is a precursor of the 16S complex. Its slower sedimentation may reflect a lack of certain structural proteins or RNAs. Another hypothesis is that the two HSP complexes are different, having related or different functions but containing both the HSPs, which, however, turn over more rapidly in the 12S complex. A comparative analysis of these two complexes should enable one to choose between the two hypotheses. Although it is clear that the prosomes and HSP complex are different, the 29-kDa protein seems to be present in both complexes. Whereas the systematic analysis of the sucrose gradient fractions shows that the two complexes are clearly


VOL. 9. 1989 1

2 3

CSK CHO A SOL W

1 2 3

2679

MAT

_"

a

hsp

P29

CH R C HR CH R CH R

B A FIG. 6. Identification of the prosomal protein p29K in the purified 16S HSP complex. Prosomes and the 16S HSP complex were purified as described in Materials and Methods and the legend to Fig. 4. (A) The purified complexes were trichloroacetic acid precipitated, washed in acetone, and dissolved in SDS buffer prior to fractionation by SDS-PAGE. (B) A duplicate gel was transferred onto nitrocellulose paper and reacted with the anti-p27K MAb. Lanes: 1, prosomes from normal cells; 2, 16S HSP complex from heat-shocked cells; 3, preexisting 16S complex from normal cells.

separable, the anti-p27/29K prosomal antibody (prepared against duck prosomes) recognized an antigen in the 16S region as well as in the 19S prosomes, as shown by immunoblotting. This could be due to contamination of the prosomes in the 16S region. However, the prosomal MAb immunoprecipitated the four low-molecular-weight HSPs, and furthermore, it recognized an antigen in the CsCI density gradient-purified HSP complex. This suggests that this prosomal protein is truly associated with the HSP complex. It is very interesting that the p29K antigen is abundant in the 4S region of the gradient in preparations from heatshocked cells as compared with those from normal cells; this suggests that in heat shock, some of the p29K protein is released from the preexisting prosome particles. Indeed in all conditions and cells tested thus far, a prosomal protein was never found outside the particle (21, 22, 32). On the basis of these data, one might even consider the possibility that the prosomal p29K antigen dissociates from the prosomes to be transferred to the HSP complex in formation. The functions of the prosomes are still unknown. In duck erythroblasts, they are associated with repressed mRNPs (42), and they have been shown to interact with mRNA, inducing inhibition of their translation (1). In addition to this biological activity, a variety of enzymatic functions have been described which are seemingly associated with particles of a similar kind (5, 9, 19). The presence of the prosomes on intermediate filaments (22) as well as the differential cytolocalization of specific prosomal antigens according to cell type and stage of differentiation opens a totally new perspective as to their function (35), and it was suggested that specific prosomes might accompany specific mRNA populations on the intermediate filaments of specific cells (40). The results shown here indicate that the heat-shocked Drosophila cells might be a good tool for the study of prosome function, since most preexisting mRNAs are

B

SOL

CSK CHO MAT

-29K CH RC HR CHR CHR FIG. 7. Small HSP and the 29K prosomal protein collapse onto the nucleus during heat shock. Following heat shock and recovery as described in the legend to Fig. 3. cells were fractionated by using the protocol of Lenk and Penman (28) as described in Materials and Methods. Identical amounts of material were separated by electrophoresis. transferred onto nitrocellulose paper, and reacted with the anti-p27K MAb. SOL. Triton X-100 soluble fraction; CSK, cytoskeleton fraction: CHO. chromatin fraction, MAT, nuclear matrix; C. control cells labeled for 18 h at 23° C; H. heat-shocked cells labeled for 2 h at 37° C; R, recovery for 6 h in cold medium after 2 h of labeling at 37° C. (A) Autoradiography of the blot. (B) Same blot reacted with the anti-p27K prosomal MAb.

shifted to the untranslated cell compartment. No modification in the protein composition of the prosome was observed after heat shock. However, the labeling of the HSP in the 16S complex was dramatically modified under stress conditions: it should be noted that a 16S complex is present under normal conditions also. This result is in favor of a precise function of these newly synthesized proteins, which become abundant in this complex upon heat shock and thus might modulate its function. Alternatively, one might speculate that in heat shock the physical, possibly cytoskeletal, system in which the HSPs are involved has to be modified and thus must be reconstituted de novo by using newly synthesized constituents. The collapse of the cytoskeletal system carrying the HSP onto the nucleus is indicative of such an event (27). Heat shock, as well as other stress conditions, induces profound modifications in the cell translation system. Early studies on heat shock in HeLa cells carried out by Penman (36) and in our own laboratory (54) showed that (i) polyribosomes fully dissociate at 42° C within 7 min and reform at high temperatures within 20 min at a different level and (ii) mRNA synthesis continues but ribosome formation is stopped, and ribosomal proteins, in particular, are no longer formed. Heat shock implies, therefore, a reprogramming of protein synthesis, which has been extensively studied in Drosophila cells (15, 30; for a review, see reference 41). Furthermore, prosomes (and thus mRNPs) are associated with the intermediate filaments of the cytoskeleton (22) as was suggested for the HSPs under certain stress conditions (27). One might suggest, therefore, since mRNA and

2680


well as the HSPs and the cytoskeleton, are all somehow linked, that a dramatic reprogramming of protein synthesis entails an important reconstruction of the system carrying translated as well as repressed mRNA. This system might transport the mRNA to precise sites of expression (26). Further studies will be necessary to elucidate the structural and functional interrelation of mRNPs and prosomes on the one hand and of the cytoskeleton and the HSP complex on the other.

prosomes, as

ACKNOWLEDGMENTS

We are grateful to H.-P. Schmid for helpful discussion, to G. Brawerman and A. M. Courgeon for critical reading of the manuscript, to Chantal Cuisinier and Richard Schwartzmann for help in the preparation of the manuscript, and to Michele Huesca for technical assistance. C. Martins de Sa and M.-F. Grossi de Sa were supported by a fellowship from the Brazilian Conselho Nacional de Desenvolvimento Cientifico e Technologico, and E. Rollet was supported by a fellowship from the Association pour la Recherche sur le Cancer. This research was supported by grants to K.S. from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the Fondation pour la Recherche Medicale Francaise, and the Ligue Francaise contre le Cancer, by a grant from the Association pour la Recherche sur le Cancer to M.B.-B., and by a grant to R.T. from the Fonds de la Recherche en Sante du Quebec. LITERATURE CITED 1. Akhayat, O., A. A. Infante, D. Infante, C. Martins de Sa, M.-F. Grossi de Sa, and K. Scherrer. 1987. A new type of prosome-like particle, composed of small cytoplasmic RNA and multimers of 21-kDa, inhibits protein synthesis in vitro. Eur. J. Biochem. 170:23-33. 2. Arrigo, A.-P., and C. Ahmad-Zadeh. 1981. Immunofluorescence localization of a small heat shock protein (hsp 23) in salivary gland cells of Drosophila melanogaster. Mol. Gen. Genet. 184: 73-79. 3. Arrigo, A.-P., J.-L. Darlix, E. W. Khandjian, M. Simon, and P. F. Spahr. 1985. Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins. EMBO J. 4:399406. 4. Arrigo, A.-P., S. Fakan, and A. Tissieres. 1980. Localization of the heat-shock induced proteins in Drosophila mnelanogaster tissue culture cells. Mol. Gen. Genet. 78:86-103. 5. Arrigo, A.-P., K. Tanaka, A. L. Goldberg, and W. Y. Welch. 1988. Identity of the 19S "prosome" particle with the large multifunctional protease complex of mammalian cells (the proteasome). Nature (London) 331:192-193. 6. Atkinson, B. G., and D. B. Waldern. 1985. Changes in eukaryotic gene expression in response to environmental stress. Academic Press, Inc., New York. 7. Bensaude, O., L. Babinet, M. Morange, and F. Jacob. 1983. Heat shock protein, first major products of zygote gene activity in mouse embryo. Nature (London) 305:331-333. 8. Bochkareva, E. S., N. M. Lissin, and A. S. Girshovich. 1988. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature (London) 336:254257. 9. Castano, J. G., R. Ornberg, J. G. Koster, J. A. Tobian, and M. Zasloff. 1986. Eukaryotic pre-tRNA 5' processing nuclease: copurification with a complex cylindrical particle. Cell 46:377387. 10. Cleveland, R. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. 11. Collier, N. C., and M. J. Schlesinger. 1986. The dynamic state of heat shock proteins in chicken embryo fibroblasts. J. Cell Biol. 103:1495-1507.

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erythroblasts. Mol. Biol. Rep. 13:35-44. 48. Tomek, W., G. Adam, and H.-P. Schmid. 1988. Prosomes, small cytoplasmic RNP particles, contain glycoproteins. FEBS Lett. 239:155-158. 49. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 50. Velasquez, J. M., and S. Lindquist. 1984. Hsp 70: nuclear concentration during environmental stress and cytoplasmic storage during recovery. Cell 36:655-662. 51. Vincent, A., 0. Akhayat, S. Goldenberg, and K. Scherrer. 1983. Differential repression of specific mRNA in erythroblasts cytoplasm: a possible role for free mRNP particles. EMBO J. 2: 1869-1876. 52. Vincent, A., 0. Civelli, K. Maundrell, and K. Scherrer. 1980. Identification and characterization of the translationally repressed cytoplasmic globin messenger ribonucleoprotein particles from duck erythroblasts. Eur. J. Biochem. 112:617-633. 53. Vincent, M., and R. M. Tanguay. 1982. Different intracellular distribution of heat-shock and arsenite induced proteins in Drosophila KC cells. J. Mol. Biol. 162:365-378. 54. Warocquier, R., and K. Scherrer. 1969. RNA metabolism in mammalian cells at elevated temperature. Eur. J. Biochem. 10: 362-370. 55. Yost, J. H., and S. Lindquist. 1986. RNA splicing is interrupted by heat shock and is reversed by heat shock proteins synthesis. Cell 45:185-193. 56. Zimmerman, J. L., W. Petri, and M. Meselson. 1983. Accumulation of a specific subset of D. melanogaster heat shock mRNA in normal development without heat shock. Cell 32:1161-1170.