expression (see reviews in Atkinson and Walden, 1985; Craig,. 1985; Burdon, 1986). These changes typically occur at two levels: 1) the new and/or enhanced ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1990 by The American Society for Biochemistry
Vol. 265, No. 24, Issue of August 25, pp. 14156-14162, 1990 Printed in U.S.A.
and Molecular Biology. Inc.
Ferritin Is a Translationally of Avian Reticulocytes*
Regulated Heat Shock Protein (Received for publication,
Burr G. Atkinson+, From the Ceil Science London, Ontario N6A
Timothy Laboratories, 5B7, Canada
W. Blaker, Department
Jeff Tomlinson,
of Zoology,
Heat-shocked avian reticulocytes exhibit enhanced synthesis of a >450-kDa protein. Biochemical, immunochemical, and visual criteria were used to identify this protein as the iron storage protein ferritin. The 21-kDa ferritin subunits synthesized during heat shock are similar in size and p1 to the subunits that are constitutively synthesized. The 2-6-fold heat shockinduced increase in ferritin synthesis appears to be regulated at the translational level as it is insensitive to actinomycin D. Northern and dot-blot hybridization analyses of cytoplasmic RNAs with avian H-ferritin cDNA fragments support the contention that the heat shock stimulation of ferritin synthesis is translationally regulated. These latter studies demonstrate that the heat shock-induced synthesis of ferritin does not involve a change in the amount of total cytoplasmic ferritin mRNAs, but rather appears to entail a translocation of cytoplasmic H-ferritin mRNAs from a polyribosome-free, translationally repressed state to a polyribosome-associated, translationally active state. These results suggest that thermally stressed aviau reticulocytes have a critical and functional need for the synthesis of additional ferritin and that its enhanced synthesis, unlike the new and/or enhanced synthesis of the well-established avian heat shock proteins, is regulated wholly at the translational level.
Eukaryotic cells respond to rapid elevations in temperature (heat shock) by expeditious changes in their pattern of gene expression (see reviews in Atkinson and Walden, 1985; Craig, 1985; Burdon, 1986). These changes typically occur at two levels: 1) the new and/or enhanced transcription of a small family of heat shock genes and 2) the preferential translation of the mRNA transcripts from these genes. In previous reports, we demonstrated that quail reticulocytes retain the capacity to express a complete heat shock response in vitro as well as in situ (Atkinson et al., 1986; Atkinson and Dean,
1985; Dean and Atkinson, 1985). Our earlier studies revealed that in addition shock proteins
to the new and/or enhanced synthesis of heat (Hsps)’ with relative molecular masses (A&)
of 90,000,70,000 and 26,000, there is also enhanced synthesis of a protein with an M, greater than 450,000 (Atkinson et al., * This work was supported by funds provided (to B. G. A.) from the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom all correspondence should be addressed. ’ The abbreviations used are: Hsps, heat shock proteins; SDS, sodium dodecyl sulfate; kb, kilobase; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.
The
University
February 22, 1990)
and Rob L. Dean of Western
Ontario,
1986). Unlike the other Hsps (as defined by Schlesinger (1986)) of quail reticulocytes, the heat shock-induced enhanced synthesis of this high molecular weight protein appeared to be insensitive to actinomycin D (Atkinson et al., 1986). The possibility that heat shock affected the synthesis of this protein wholly at the translational level prompted us to pursue the characterization and identification of this protein. The results reported herein extend our previous results on the heat shock response of quail reticulocytes and provide biochemical, immunochemical, and electron microscopic evidence to support our conclusion that the heat shock-induced enhanced synthesis of this >450,000-dalton protein is translationally regulated and that this protein is ferritin, a major intracellular iron sequestering and iron storage protein (Theil, 1987; Munro and Linder, 1978). Although the control of ferritin synthesis by iron (Theil, 1987; Munro and Linder,
1978) has been well documented, very few observations on the regulatory mechanism governing the expression of ferritin genes by other agents have been reported (Dickey et al., 1988; Torti et al., 1988; Cox et al., 1988). EXPERIMENTAL
PROCEDURES
Heat Shocking, Radioactive Labeling, and Lysing of Reticulocytes Isolated from Anemic Quail-Adult (3-month-old) Japanese quail (Coturnix coturnir japonica, reared in the Department of Zoology at The University of Western Ontario) were made anemic with phenylhydrazine (Spohr et al., 1972; Dean and Atkinson, 1985), their blood was collected and red blood cells were isolated from it by pelleting
them through
Ficoll-Paque
(Pharmacia
Fine Chemicals, Uppsala,
as previously described (Dean and Atkinson, 1985; Atkinson et al., 1986). Washed reticulocytes were suspended in methionine-free minimal essential medium (GIBCO) to a concentration of 0.3-1.2 X lo6 cells/ml. Cell cultures were incubated at temperatures ranging from 37 to 45 “C and labeled with 60 &i/ml of L-[35S]methionine (1196.3 Ci/mmol; Du Pont-New England Nuclear) for various periods of time (see figure legends for details). In some cases, the original culture medium was supplemented with various concentrations of actinomycin D (see figure legends for details). After labeling, cells were pelleted at 100 x g for 10 min, washed in cold saline. and nelleted. Pelleted cells were lysed either hypotonically (Granger kt aZ.,-1982) in 4 volumes of 10 n& triethanoiamine-HCi, pH 7.4, containing 10 mM NaCl, 1 mM MgCl,, 5% p-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride, or in 4 volumes of a buffer (10 mM Tris-HCl. nH 7.2. 150 mM NaCl. 5 mM M&l*, 2 mM EGTA, 6.25 mM dithiothieitol, and 1 mM ph&ylmethyisuifonyl fluoride) containing 1% Triton X-100 according to the method of Blikstad and Lazarides (1983). In either case, lysates were centrifuged at 200 X g for 10 min, and the supernatants were used for subsequent analyses. In some cases, the sunernatants were heated to 75-80 “C for 20 min, cooled on ice for 30 min, centrifuged at 5000 x g for 30 min, and the supernatants were transferred to separate tubes for immediate use or storage at -70 “C. In other cases, since resistance to proteinaseK digestion is characteristic of ferritin (Atkinson et al., 1989; Nichol and Locke, 1989), proteinase-K (Sigma) was prepared in 80 mM TrisHCl, pH 6.8, and added to the supernatants so that the final amount of enzyme/p1 of sample was 0.2 fig (ie. 1 pg of proteinase-K/80 rg of Sweden)
14156
Ferritin
Is a Heat Shack Protein
protein). The enzyme-treated supernatants were incubated in capped tubes at 37 “C for 15 min on a shaker bath. After the 37 “C incubation, the samnles were heated at 75-80 “C for lo-20 min (to inactivate and precipitate the denatured enzyme (proteinase-K is inactivated at T 1 65 *C; Kersey, 1987)) and cooled on ice for 30 min. The cooled samples were centrifuged (5000 x g/20 min), and the resulting clear supernatants were concentrated in type CF50A Centriflo membrane cones (Amicon Corp., Danvers, MA) and either used directly or stored at -70 “C (Atkinson et al., 1989). Gel Eleetrophoresis and Immunoblotting-One-dimensional gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was performed according to the method of Laemmli (1970) except that the separating gel consisted of either a 7.5-17.5% or 5-15% polyacrylamide gradient overlaid with a 3% stacking gel. Samples were adjusted to contain 2% SDS and clarified by centrifugation in a Beckman microfuge (Beckman Instruments, Spinco Div., Palo Alto, CA) prior to electrophoresis. Two-dimensional analysis of the proteins in the cell lysates followed, with minor modifications, the method developed bv O’Farrell(1975). Nonidet P-40 (BDH). urea (to a tinal concentrat&n of 9 M),‘ and ‘ampholines (pH range 3.5-16, LKB Instruments Inc., Rockville, MD) were added to the samples just prior to isoelectric focusing. The second dimension polyacrylamide gel slabs consisted of either a 7.5-17.5% or 5515% polyacrylamide gradient overlaid with a 3% polyacrylamide stacking gel. To characterize the isoelectric point (~1). mass (M,) of the electrophoretically _ and relative molecular separated polypeptides, marker proteins of known p1 and M, were electrophoretically separated on the same gels (Atkinson et al., 1986). Molecular weight marker polypeptides consisted of standards from a low molecular weight calibration kit purchased from Pharmacia Fine Chemicals, as well as ferritin and apoferritin (Sigma). In some cases, the gel-separated, [35S]methionine-labeled proteins were electrophoretically transferred to nitrocellulose membranes and processed for enzyme immunoassay, using rabbit anti-horse ferritin as the primary antibody (Miles Laboratories, Naperville, IL; shown to cross-react with ferritins from a variety of sources, including avians (Theil and Brenner, 1981; Atkinson et al., 1989)) and goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) as the secondary antibody (Atkinson et al., 1989). The relative amount of ferritin in these immunoblots was determined by laser densitometry (Ultroscan Laser Densitometer; LKB Instrument, Inc.), and the immunostained ferritin bands were excised from the nitrocellulose and quantitatively assessed for radioactivity (Atkinson et al., 1989). Extraction, Staining, and FEworographic Annlysis of Electrophoretically Separated Proteins-After electrophoresis, the proteins in the gels were either stained directly with Coomassie Brilliant Blue G (Sigma) or first stained for iron (Chung, 1985), photographed, destained, and subsequently stained with Coomassie Brilliant Blue G. The Coomassie Blue-stained gels were photographed, impregnated with 2,5-diphenyloxazole, dried down onto Whatman 3MM filter paper (Bonner and Laskey, 1974) with a Bio-Rad model 224 Gel Slab Dryer, and, as described elsewhere (Atkinson, 1981), fluorograms were prepared from them. Stained gels and fluorograms of these gels were scanned on an Ultroscan Laser Densitometer (LKB), and holoand/or apo-ferritin, and ferritin subunits were subsequently extracted from corresponding areas of the fluorographed gels and assessed for radioactivity (Atkinson, 1981). In some cases, the electrophoretically separated proteins were detected in the gels bv briefly immersing the gels in cold 0.25 M KC1 (Hager and Burgess, i980). Bands con&ring-the visualized proteins were excised from the gels, and the proteins were extracted from the gel by electrodialysis (Bhown et al., 1980) using an Isco Sample Concentrator (model 1750; Isco Instrumentation Specialities Co.) and visualized by electron microscopy, or assessed for iron (Drysdale and Munro, 1965), protein (Comings and Tack, 1972), and radioactivity (Dean and Atkinson, 1985; Atkinson et al., 1986). Isokxtion of Total Cyto&smic and Polyribosome-associated RNAsControl and heat-shocked reticulocyteswere lysed in an RNA extraction buffer containing 0.14 M NaCl. 1.5 mM Me& 10 mM Tris-Cl. pH 8.6, 0.5% Nonidet P-40, and i mM dithiithreitol. Nuclei and unlysed cells were removed from the lysates by low speed centrifugation, and the supernatants were used as the source of both total cytoplasmic RNAs and polyribosome-associated RNAs. Total cytoplasmic RNAs were extracted from the supernatants with guanidinium thiocyanate as described by Sambrook et al. (1989). A portion of the supernatants was layered over a 1.65 M sucrose cushion made up in RNA extraction buffer, centrifuged at 50,000 rpm (type 50 rotor, Beckman Instruments) for 3.5 h at 4 “C in a Beckman Ultracentrifuge (model L8-70M; Beckman Instruments), and the polyri-
of Avian
Reticulocytes
14157
bosome pellet was collected. Polyribosome-associated RNAs were extracted from the polyribosomes with guanidinium thiocyanate as described for cells by Chomczynski and Sacchi (1987). Northern and Dot-blot Hvbridizations-For Northern blotting, 10 pg samples of RNA were separated by electrophoresis on 1% agaroseformaldehyde denaturing gels as outlined by Maniatis et al. (1982). The electrophoretically separated RNAs were transferred to Gene Screen (Du Pont-New England Nuclear) by capillary elution as outlined in the manufacturer’s instructions. In some cases, 1,5, or 10 pg of RNA were dot-blotted onto GeneScreen using a Bio-dot microfiltration apparatus (Bio-Rad). Probes used for hybridization included a 4.2-kb EcoRI-BamHI DNA frazrnent from nlasmid DBIBR~~ containing a chicken B-globin gene (Dolan et al., i983), a- i.8-kb Hind111 DNA fragment-from plasmid pC1.8 containing a chicken Hsp 70 gene (Morimoto et al., 1986), a 0.73-kb EcoRI cDNA fragment from plasmid pGEMI-56DcDNA-20A containing the entire coding region and the 3’-untranslated region of a chicken red blood cell H-ferritin subunit (Stevens et al., 1987), and a 0.60-kb PstI DNA fragment from a chicken ubiquitin cDNA (Bond and Schlesinger, 1985). All of the DNA inserts used as probes in the hybridization procedures were digested from the vectors with restriction enzymes purchased from Bethesda Research Laboratories (BRL) or Pharmacia LKB Biotechnology Inc., and electrophoretically separated on agarose gels. The gel-separated DNA fragments were excised from the gels, melted, purified using Geneclean (BIO.101 Inc., LaJolla, CA), and labeled with [a-32P]dCTP to a specific activity of -1 X 10’ cpm/pg using the random primer Oligolabeling Kit from BRL. The labeled DNA fragments were chromatographically purified off Sephadex G-50 columns (Pharmacia). Hybridization was conducted in PR-800 Hvbridease chambers (Hoefer) at 42 ‘C for 24 h in the presence of 50% formamide and 10% dextran sulfate, and the blots were washed to a stringency of 0.1 x SSC (standard sodium citrate) at room temperature. Following autoradiography, blots were stripped by washing in 0.1 X SSC for 3 h at 85 “C. Electron Microscopy of the >450-kDa Protein and Ferritin Purified from Quail Reticulocytes-Small drops (0.5 ~1) of an aqueous solution of electrophoretically purified >450-kDa protein or horse spleen ferritin (Sigma) were smeared across Formvar-coated, carbon-shadowed copper grids with a piece of filter paper and allowed to air dry. Such unstained preparations were viewed directly. Other preparations were negatively stained with sodium phosphotungstate (Locke and Huie, 1980) or were stained with bismuth at pH 9 by the methods of Nichol and Locke (1989). Grids were viewed on a Philips EM 200 at 80 kV. RESULTS
The Heat Shock Response of Quail Reticulocytes-When quail reticulocytes are subjected to brief elevations in temperature, they exhibit new and/or enhanced synthesis of polypeptides with M, values of approximately 26,000, 70,000, 90,000, and >450,000 (Fig. 1). In this study, we focused our attention on a putative Hsp with an M, > 450,000. Although this polypeptide is constitutively synthesized in reticulocytes, its synthesis appears to be enhanced in lysates from reticulocytes incubated for 2 h at temperatures greater than 40 “C (Fig. L4). The electrophoretic mobility of this >450-kDa protein appears to change when the 200 X g Triton X-100 supernatants from reticulocyte lysates are boiled for 2 min in the presence of SDS; after this treatment the >450-kDa protein becomes less apparent in the fluorograms, and a concomitant increase in the radioactivity of a polypeptide of approximately 21 kDa is observed (Fig. 1B). Churacterization of the >450-kDa Hsp as Ferritin-Native ferritin (Mr > 450,000) is normally heat stable (i.e. it is not precipitated or separated into its subunits by a lo-min incubation at 75-80 “C in aqueous solution at pH 7.0 (Hoffman and Harrison, 1963; Listowsky et al., 1972)), is insensitive to a number of proteolytic enzymes including proteinase-K (Coffey and de Duve, 1968; Atkinson et al., 1989; Nichol and Locke, 1989), generally contains iron (i.e. holo-ferritin; Theil, 1987), displays a characteristic appearance in the electron microscope (Munro and Linder, 1978; Nichol and Locke, 1989), and can be separated into its -21-kDa subunits by
Ferritin
Is a Heat Shock Protein of Avian Reticulocytes A
~16~
M,
a
--e
. 4
90 70
.
26
12 c-
37
B
-
40
42
44
*
-
46'C
1-*
4 >450
m
'duiiic
I-IS -A
21 HS C +A
FIG. 1. The synthesis of a >450-kDa protein appears to be enhanced by heat shock. The fluorograms shown in this figure are from SDS-polyacrylamide gel electrophoretically separated, L-(~?S] methionine-labeled proteins synthesized by quail reticulocytes incubated (panel A) for 2 h at the temperatures indicated in the lower part of this pane1 or (panel B) for 2 h at 37 “C (C) or 45 “C (HS). The samples used in this figure were obtained from low speed supernatants of Triton X-100 lysates. They were adjusted to contain 2% SDS and contain equal amounts of protein (80 pg). The fluorogram shown in panel A illustrates that cells maintained for 2 h at temperatures above 40 “C show enhanced synthesis of a protein with a molecular mass of >450,000 as well as the previously reported -26-, 70-, and SO-kDa Hsps (Atkinson and Dean, 1985). The fluorograms shown in panel I3 compare the distribution of the “‘S-labeled, electrophoretically separated proteins from samples which were adjusted to contain 2% SDS and subject to electrophoresis directly (-A) or boiled for 2 min prior to electrophoresis (+A). Fluorograms of the proteins electrophoretitally separated from lysates boiled in SDS show a sharp decrease in abundance of the newly synthesized >450-kDa protein and a concomitant increase in a 21-kDa protein, suggesting that boiling in SDS separates the >450-kDa protein into 21-kDa subunits. Arrowheads on the right side of these panels indicate proteins which appear to exhibit enhanced synthesis at elevated temperatures. The relative molecular masses (M,) shown in this figure and subsequent figures were established by the inclusion of marker proteins with known M, values.
boiling in the presence of SDS and P-mercaptoethanol (Schaefer and Theil, 1981; Atkinson et al., 1989). Figs. 2 and 3 demonstrate that all of these properties are exhibited by this
d
M,x1c3
.W-
3
4
a:450
5
12
3
4
5
FIG. 2. The >450-kDa protein from lysates of heat-shocked quail reticulocytes has biochemical properties identical to ferritin and, like ferritin, consists of 21 kDa subunits. The SDS-polyacrylamide gel electrophoretic separations shown in panel A are from samples of heat-shocked reticulocytes which were lysed in Triton X-100, adjusted to contain 2% SDS, and subjected directly to electrophoresis. They demonstrate that the >450-kDa protein (fluorogram shown in lane I), like ferritin, contains iron (stained gel shown in lane 2), and immunoreacts with ferritin antibodies (immunoblot shown in lane 3). They also show that when Triton X-100 lysates are heated at 75 “C for 20 min and the precipitated protein pelleted by centrifugation, the >450-kDa protein, like ferritin, remains in the supernatant (fluorogram from a supernatant shown in lane 4), and that when lysates are treated with proteinase-K, the >450-kDa protein, like ferritin, is not digested (fluorogram shown in lane 5). The samples used in the electrophoretic separations shown in panel B are aliquots from the same preparations used for the corresponding lanes in panel A except that prior to electrophoresis the samples were adjusted to contain 2% SDS and boiled for 2 min. The results shown in panel B demonstrate that boiling in SDS separates the >450-kDa protein into 21-kDa subunits which react with ferritin antibodies (lane 3) but, like ferritin, no longer contain iron (lane 2). In both A and B, equal amounts of protein (80 pg) were applied to lanes 1-3, and equal volumes of 10 x concentrated supernatants were applied to lanes 4 and 5. In the latter case, the supernatants were derived from l-ml aliquots of Triton X-100 lysates which had been treated with heat (75 “C for 20 min) or proteinase-K and centrifuged, see “Experimental Procedures” for details.
>450-kDa putative Hsp. In lysates from control (not shown) and heat-shocked cells, this >450-kDa protein contains iron (lane 2 in Fig. 2A), is heat stable (lane 4 in A), and is refractory toward proteinase-K activity (lane 5 in A). When supernatants from untreated cell lysates and from proteinase-K or heat-treated lysates of control (not shown) and heat-shocked cells are boiled in the presence of SDS and &mercaptoethanol, the >450-kDa native protein is separated into polypeptides of approximately 21 kDa (lanes 1, 3-5 in B) and does not stain for iron (lane 2 in B). In addition, this high molecular weight protein and its subunits immunoreact with ferritin antibodies (lane 3 in panels A and B). An electron microscopic comparison of negatively stained whole mounts of horse spleen ferritin and the >450-kDa polymer (electroeluted from excised gel slices of electrophoretically separated proteins from lysates of heat-shocked reticulocytes, as shown in lane 1 of Fig. 2A) show that they are similar in size (11 nm in diameter) and in appearance (Fig. 3, A and B). Finally, the identification of this putative Hsp as ferritin was substantiated by two further observations. Like horse spleen or insect ferritins (Nichol and Locke, 1989), 1) bismuth-stained preparations of the >450-kDa particles (Fig. 3C) reveal a central core with axial projections, and 2) unstained preparations (Fig. 30) show an electron dense (iron) core, 7 nm in diameter. Comparison and Quuntitution by Control and Heat-shocked
of
the Ferritins
Synthesized
Reticulocytes-Since ferritin polymers (MI > 450,000) can be composed of monomer sub-
Ferritin
Is a Heat Shock Protein of Avian Reticulocytes
FIG. 3. The >450-kDa protein from lysates of heat-shocked quail reticulocytes has an electron microscopic image identical to ferritin. Electron micrographs of negatively stained horse spleen ferritin and the >450-kDa protein (electroeluted from gels) are shown in panels A and R, respectively. Micrographs of bismuthstained and unstained >450-kDa protein preparations are shown in panels C and D, respectively.
‘r
r
20nm
A
M, x103 > *-‘
B
* 4
0
23
I
IEF m n- I
14159
21.0 18.8
D
A
C
1”s _
i
* -A-
1
If1
M,
;lD3
- D453
5
4
* COtl.
u-l
HS
-0
A A I A 5 9 52
->450
4 PH
FIG. 4. The >450-kDa ferritin from control and heatshocked avian reticulocytes is composed of subunits having the same M, and p1 values. A, one-dimensional SDS-polyacrylamide gel electrophoretic comparisons of the mobility of ferritin subunits from control reticulocytes (lane 2,2 h at 37 “C) with ferritin subunits obtained from heat-shocked reticulocytes (lane 3; 2 h at 45 “C), horse spleen (lanes I and 4), and human heart (lane 5). The samples used in this gel were adjusted to contain 2% SDS, boiled for 2 min, separated by electrophoresis, and stained with Coomassie Brilliant Blue G. B, fluorograms prepared from two-dimensional (isoelectric focusing (IEF)/SDS) polyacrylamide gel electrophoretic separations of ferritin subunits from control (Con.) and heat-shocked (HS) quail reticulocytes. The isoelectric points (pl) of the different isoforms were determined as described elsewhere (Saleem and Atkinson, 1976). units having slightly different M, and p1 values in different cells and, under different physiological conditions, in the same cell (Theil, 1987; Mertz and Theil, 1983), it was necessary for us to qualitatively compare, prior to any quantitative synthetic studies, the M, and p1 values of the ferritin subunits synthesized in the presence and absence of heat shock. Our results, shown in Fig. 4, demonstrate that the ferritin subunits synthesized during heat shock are very similar, if not identical, to the ferritin subunits synthesized in the absence of heat shock, with an M, - 21,000 (Fig. 4A) and p1 values of 5.9, 5.5, 5.3, and 5.2 (Fig. 4B). To confirm that the synthesis of ferritin is enhanced by heat shock, we incubated quail reticulocytes at 37 “C for 60, 90 (not shown), and 120 min or at 45 “C for 60, 90, and 120 min, labeled them with L-[35S]methionine during the last 60 min of incubation, and lysed them with Triton X-100. Equal amounts of protein from the low speed supernatant of the lysates were adjusted to contain 2% SDS, and either subjected to electrophoresis directly, or boiled for 2 min prior to electrophoresis. Fluorograms were prepared to locate ferritin within the gel (see Fig. 5A), the ferritin and ferritin subunits
. 21 - Glchln I
I 0.5 Act
1.0 2.0 D (ug/ml)
FIG. 5. The synthesis of ferritin is enhanced 2-6-fold by heat shock and its enhanced synthesis is insensitive to actinomycin D. The fluorograms shown in these figures are from onedimensional SDS-polyacrylamide gel electrophoretic separations of equal amounts of protein (80 fig/lane) from the supernatants of Triton X-100 lysates of quail reticulocytes. The samples used in these gels were adjusted to contain 2% SDS and either subjected directly to electrophoresis (upper portion of A and B) or boiled for 2 min prior to electrophoresis (lower portion of A and B), and only those areas of the gels corresponding to the >450-kDa ferritin polymers (upper portion in panels A and B) or 21-kDa subunits (lower portion in panels A and B) are emphasized in these panels. The cells used in panel A were incubated either at 37 “C for 60 and 120 min (C) or at 45 “C for 60, 90, and 120 min (Ifs) and labeled with L-[““Slmethionine for the last 60 min of incubation. The cells used for panel B were incubated either in the absence of actinomycin D at 37 “C (C) or 45 “C (HS) or in the presence of actinomycin D (0.5, 1.0, or 2.0 rg/ml) at 45 “C (I-IS) for 120 min, and labeled in the last 60 min of incubation. Ferritin and ferritin subunits were subsequently extracted from corresponding areas of the fluorographed gels shown in A and B and assessed for radioactivity. Duplicate gels were run in parallel, blotted, and immunoreacted with ferritin antibodies (see “Experimental Procedures”). The relative amount of ferritin in the immunoblots was evaluated by laser densitometry and the immunostained ferritin bands were excised from the nitrocellulose and assessed for radioactivity. Determination of the specific activity of ferritin and ferritin subunits indicates that heat shock stimulates the synthesis of ferritin 2-6-fold (from -70 to 90 cpm/pg of ferritin in control to -200-600 cpm/pg of ferritin in heat-shocked cells), and that the heat shockstimulated synthesis of ferritin still occurred in the presence of concentrations of actinomycin D (2 pg/ml) sufficient to reduce total RNA synthesis by -94% and abolish the synthesis of the 90-, 70-, and 26-kDa Hsps (Atkinson and Dean, 1985; Atkinson et al., 1986).
14160
Ferritin
Is a Heat
Shock
Protein
were excised from the gel and the radioactivity assessed by liquid scintillation counting (Atkinson, 1981). Duplicate gels, run in parallel, were blotted and immunoreacted with ferritin antibodies (Atkinson et al., 1989). The amount of ferritin and ferritin subunits was assessed by laser densitometry, and the immunostained ferritin bands were excised from the nitrocellulose and assessed for radioactivity. Calculation of the specific activity (cpm/pg ferritin) indicates that heat shock stimulates the synthesis of ferritin 2-6-fold (from -70 to 90 cpm/ Fg ferritin in control, to -200-600 cpm/pg ferritin in heatshocked cells). Moreover, when we preincubated control and heat-shocked reticulocytes in concentrations of actinomycin D previously shown to effectively block both RNA synthesis and the new and/or enhanced synthesis of the other quail reticulocyte Hsps (2 pg/ml; see Atkinson and Dean, 1985; Atkinson et al., 1986), the 2-6-fold heat shock-stimulated synthesis of ferritin still occurred (Fig. 5B). Relative Abundance of Ferritin mRNAs among the Total Cytoplasmic and Polyribosomal-associated mRNAs in Control and Heat-shocked Reticulocytes-To clarify our interpretation of the apparent differences in the synthesis of ferritin in control and heat-shocked reticulocytes, we used a cDNA for chicken red blood cell H-ferritin (only a single copy of this gene is found in the chicken genome (Stevens et al., 1987)) to assess the relative abundance of total H-ferritin mRNAs in the cytoplasm as well as the relative abundance of H-ferritin mRNAs associated with polyribosomes in control and heatshocked quail reticulocytes. We subsequently hybridized the same blots with DNA fragments encoding sequences complementary to chicken @-globin and to two well-established avian heat shock proteins, Hsp 70 and ubiquitin. Hybridization of these DNA fragments to Northern blots of polyribosomeassociated RNAs (Fig. 6A) indicates the sizes of the mRNAs encoding these proteins in control and heat-shocked cells, and suggests that heat shock appears to change the relative amounts of these mRNAs associated with polyribosomes. Dot blots of total cytoplasmic RNAs hybridized with these DNA fragments (Fig. 6B) demonstrate that the relative amounts of total cytoplasmic ferritin and globin mRNAs do not change as a result of heat shock, whereas, the relative amount of total cytoplasmic Hsp 70 and ubiquitin mRNAs are increased in heat-shocked cells. An assessment of the relative abundance of these mRNAs on polyribosomes in control and heat-shocked cells (Fig. 6C) reveals that the amount of globin mRNA associated with polyribosomes is markedly decreased in heat-shocked cells while the amounts of ferritin, Hsp 70, and ubiquitin mRNAs associated with polyribosomes substantially increase. The apparent mobilization of ferritin mRNAs from a presumably inactive cytoplasmic form to an active polyribosome-associated form as a result of heat shock, as demonstrated in Fig. 6, B and C, supports our contention that ferritin synthesis is enhanced by heat shock and that its enhanced synthesis is partially, if not wholly, regulated at the translational level. DISCUSSION Ferritins are iron sequestering and iron storage proteins found in virtually all cells (Theil, 1987). Iron administration to animals, plants, or cells in culture stimulates the synthesis of ferritin subunits (Theil, 1987; Munro and Linder, 1978). The iron-induced enhanced synthesis of ferritin is primarily regulated at the post-transcriptional level and involves a mobilization of a cytoplasmic pool of free, inactive ferritin mRNAs on to polyribosomes (Zahringer et al., 1976; Aziz and Munro, 1986). The translational regulatory machinery involved in the iron-induced activation of these mRNAs in-
of Auian
Reticulocytes
A Globin C
Ferritin C HS
HS
Ubiq.
C
HSP
70
C
HS
HS
Kb
I)
l
2.6
l
1.6
1 . 1.1 4 0.9
$
-0
B
C Total Cytoplasmic RNA
Polyribosomal Associated RNA
0
FIG. 6. Northe] ferritin, @-globin, polyribosome-associated
l
Globin Ferritin
0
;
Kbq.70
C
HS and dot-blot ubiquitin, and mRNAs.
l
l
l
l
: C hybridization Hip 70 total
HS analysis cytoplasmic
of Hand
The autoradiograms shown in panel A are from Northern blots of polyribosome-associated RNAs from control (C, 2 h at 37 “C) and heat-shocked (HS, 2 h at 45 “C) reticulocytes hybridized with 3ZP-labeled DNA fragments encoding transcribed portions of mRNAs for fl-glohin, H-ferritin, ubiquitin (Ubiq.), and Hsp 70 as described under “Experimental Procedures.” The relative size (kb) of the individual mRNAs are shown on the right and were determined from ethidium bromide-stained RNA markers separated on the same gels. Aliquots (5 pg) of total cytoplasmic RNAs (panel B) and polyribosome-associated RNAs (panel C) from control (C) and heat-shocked (HS) reticulocvtes were dotblotted and hybridized with labeled DNA fragments fir P-globin, Hferritin, ubiquitin, and Hsp 70. The autoracliograms in panels B and C are representative of results obtained from hybridizations of at least six different sets of dot blots, and they show that although the amount of cytoplasmic ferritin mRNA is the same in control and heat-shocked cells (panel B), heat shock causes an increase in the amount of ferritin mRNAs associated with polyrihosomes (panel 0. eludes an iron-responsive element of 28 highly conserved nucleotides in the 5’-untranslated region of ferritin mRNAs (Hentze et al., 1987; Aziz and Munro, 1987; Stevens et al., 1987), and a ferritin repressor protein which inactivates ferritin mRNAs by binding to the iron-responsive element (Leibold and Munro, 1988; Rouault et al., 1988; Walden et al., 1988). Increased intracellular levels of chelatable iron (Rogers and Munro, 1987; Mattia et al., 1989) or other forms of iron, such as hemin (Lin et al., 1990), have been postulated to interact with the ferritin repressor protein, either directly or indirectly, and mediate the derepression of ferritin mRNAs. In this study, we demonstrate that heat-shocked quail reticulocytes exhibit new and/or enhanced synthesis of Hsps
Ferritin
typical of quail cells (Atkinson, 1981; Dean and Atkinson, 1985; Atkinson et al., 1986) as well as enhanced synthesis of a protein with an M, > 450,000. This latter protein is identified as ferritin by biochemical, immunochemical, and visual properties and appears to consist primarily of H-ferritin subunits. The heat shock induction of ferritin synthesis in reticulocytes is similar to that established for the iron induction of ferritin synthesis in a number of cell types in that its enhanced synthesis appears to be partially, if not wholly, regulated at the post-transcriptional level. As shown here, the heat shock-induced synthesis of ferritin is insensitive to actinomycin D and appears to involve a translocation of Hferritin mRNAs from a polyribosome-free, translationally repressed state to a polyribosome-associated, translationally active state. Unlike heat shock and iron, which appear to act by enhancing the translation of ferritin mRNA, an unidentified factor present in reticulocyte lysates, but not in wheat germ lysates, represses the translation of ferritin mRNA (Dickey et al., 1988). Other agents, such as tumor necrosis factor-a (Torti et al., 1988) and thyrotropin (Cox et aZ., 1988), appear to regulate ferritin synthesis by enhancing the transcription of ferritin mRNA. The actual mechanism by which heat shock (stress) affects the induction of specific transcriptional and translational changes in cells is not known. However, results from a number of studies support the hypothesis that heat stress initiates the generation of denatured and abnormal proteins which are proposed to directly or indirectly mediate the new and/or enhanced expression of the heat shock proteins (Kelley and Schlesinger, 1978; Hightower, 1980; Kelley and Schlesinger, 1982; Goff and Goldberg, 1985; Munro and Pelham, 1985; Ananthan et al., 1986; Edington et al., 1989). Regardless of what the case may be for affecting a general heat shock response, we demonstrate that the enhanced synthesis of ferritin detected in heat-shocked reticulocytes is a direct result of the derepression and translocation of ferritin mRNAs. The mechanism by which inactive, free cytoplasmic ferritin mRNAs are derepressed most likely involves the destabilization or removal of the ferritin repressor protein from the ironresponsive elements (Leibold and Munro, 1988, Rouault et al., 1988, Walden et al., 1988). Thus, in keeping with the abnormal and/or denatured protein hypothesis mentioned above, we propose that heat stress affects the induction of enhanced ferritin synthesis by denaturing (or destabilizing) the ferritin repressor protein or by denaturing some intracellular iron-containing proteins, such as hemoglobin. In the latter case, the denaturation of hemoglobin would lead to the release of iron or hemin-like products into the intracellular milieu which, in turn, could destabilize or remove the ferritin repressor protein, derepress the ferritin mRNAs, and promote ferritin synthesis. Whatever the case, the established, important intracellular function that ferritin normally plays in protecting a cell from the adverse affects of free iron (Munro and Linder, 1978; Theil, 1987), coupled with its enhanced synthesis during heat shock, as reported herein, suggests an augmentative functional need for it in red blood cells during thermal stress. Acknowledgments-We wish to thank Dr. J. the nlasmic DBIBRIB. Dr. P. W. Stevens for pGEMI-56D:cDNA 2bA, Dr. R. Morimoto for pC1.8, and Dr. M. J. Schlesinger for supplying also wish to acknowledge our appreciation to excellent electron micrographs. This work possible without their generosity.
14161
Is a Heat Shock Protein of Avian Reticulocytes
D. Engel for supplying sunulvine the ulasmid supplying the plasmid ubiquitin cDNA. We Dr. M. Locke for the would not have been
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