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Originally published In Press as doi:10.1074/jbc.M406999200 on July 16, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39999-40006, September 17, 2004
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Oligomerization, Chaperone Activity, and Nuclear Localization of p26, a Small Heat Shock Protein from Artemia franciscana*

Yu Sun{ddagger}, Marc Mansour, Julie A. Crack, Gillian L. Gass, and Thomas H. MacRae§

From the Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada

Received for publication, June 23, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 {alpha}-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The small heat shock proteins (sHsps),1 characterized by a conserved {alpha}-crystallin domain exhibiting an immunoglobulin-like fold bordered by variable amino- and carboxyl-terminal extensions (1-7), function as molecular chaperones. The molecular mass of sHsps ranges from 12 to 43 kDa, and most oligomerize by a multistep process often with dimers as stable suboligomeric units (8, 9), although for bovine {alpha}B-crystallin monomers may be basic building blocks (10). For sHsps such as Hsp20 (11) and Hsp22 (12), dimers are the predominant complex, and they exhibit chaperone activity. Of medical significance, {alpha}A-crystallin truncations occur in mammalian lens, suggesting a relationship between carboxyl-terminal modification and cataract (13, 14). Also, dropping either 13 or 25 carboxylterminal residues from human {alpha}B-crystallin causes myofibrillar myopathy, with modified proteins exerting dominant negative effects (15). The truncated and normal {alpha}B-crystallins appear to interact, yielding oligomers slightly smaller than those generated by wild type protein.

Assembly mechanisms and the resulting oligomers vary for sHsps from different sources (2, 4, 16-18). For example, oligomers of Hsp16.5 from the Archaea, Methanococcus jannaschii (19) and Hsp16.9 from wheat, Triticum aestivum (18), the only crystallized sHsps, are monodisperse, demonstrating well defined stoichiometry. In contrast, oligomers from other species are polydisperse and resist crystallization (3, 10, 20). sHsp oligomers exhibit rapid subunit exchange, a property that influences chaperone function (9, 11, 16, 21-25). The sHsps bind proteins in the molten globular state which are primed for aggregation and potential irreversible precipitation (26). These substrates, involved in functions from transcription to secondary metabolism, are subsequently released and refolded, activities reported to depend upon ATP-requiring chaperones such as Hsp70 (1, 21, 27-30).

Embryos of the brine shrimp, Artemia franciscana, undergo ovoviviparous and oviparous development, the former yielding nauplii and the latter encysted gastrulae or cysts (31). Cysts enter diapause (32, 33), characterized by deep reduction in metabolic activity (34) and resistance to extreme environmental stress such as long term anoxia, desiccation, and heat shock (35, 36). p26, an abundant sHsp in Artemia cysts, forms oligomers, functions as a molecular chaperone in vitro, and confers thermo-tolerance on transformed bacteria (29, 37-41), undoubtedly contributing to embryo stress tolerance. p26 enters nuclei upon synthesis in developing embryos (38) and migrates reversibly by a poorly understood mechanism into Artemia nuclei during anoxia and heat shock (42-44). Upon exposure to anoxia the internal pH of postdiapause cysts drops to about 6.6, and reversible movement of p26 into nuclei occurs in vitro upon pH reduction. Domain-specific effects on p26 oligomerization, chaperone activity, and nuclear localization were examined in this study, and the data are related to the structural/functional properties of p26 within oviparously developing Artemia embryos.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of p26 cDNAs—Truncated p26 cDNAs were generated by site-directed mutagenesis using the QuikChange Site-directed Mutagenesis kit (Stratagene, La Jolla, CA), p26-3-6-3 as template (38), and designated primers (Table I). The truncated p26-3-6-3 cDNAs were recovered from agarose gels with the GFX PCR DNA and Gel Band purification kit (Amersham Biosciences), inserted into pRSET.C, a polyhistidine-tagged (His-tagged) prokaryotic expression vector linearized by digestion with BamHI and XhoI prior to purification from agarose gels, and cloned in Escherichia coli strain BL21(DE3)PLysS (Invitrogen). Full-length and truncated p26 cDNAs were also generated previously by PCR and inserted into the T/A vector, pCRII (37). The p26 cDNA-containing pCRII vectors and the mammalian expression vector pSecCMV (Invitrogen) were digested with BamHI and XbaI, followed by electrophoresis in 1% agarose gels. p26 cDNAs were recovered with the GFX PCR DNA and Gel Band purification kit, inserted into linearized pSecCMV, and cloned in E. coli DH5{alpha} (Invitrogen). Full-length p26 cDNA (p26-full) and the cDNA fragments p26-N{Delta}60 and p26-{alpha} were excised from pSecCMV with BamHI and XbaI, inserted into pcDNA4/His.A (Invitrogen), a His tag-containing mammalian expression vector, and cloned in E. coli DH5{alpha}. All inserts were sized by restriction digestion followed by electrophoresis in agarose, and PCR fidelity was verified by DNA sequencing (DNA Sequencing Facility, Centre for Applied Genomics, Hospital for Sick Children, Toronto, ON).


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  		TABLE I
Primers for site-directed mutagenesis of p26

Truncated p26 variants were generated by site-directed mutagenesis using the primers listed below in the 5' to 3' direction. The name of each p26 variant, either cDNA or protein, is indicated in the left column. Full-length p26, not shown in the table, is termed p26-full.

 
Purification of p26 —Expression plasmids containing either full-length or truncated p26 cDNAs transformed into E. coli strain BL21(DE3)PLysS were induced by the addition of 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside (Sigma) for 5 h. The cultures were cooled on ice and centrifuged at 5,000 x g for 5 min at 4 °C. The cell pellets were washed once with extraction/wash buffer (50 mM Na3PO4, 300 mM NaCl, pH 7.5) and resuspended in 4 ml of the same buffer prior to the addition of 100 µg/ml lysozyme (Sigma), 1 mM phenylmethylsulfonyl fluoride (Sigma), 1 µg/ml pepstatin A (Sigma), and 1 µg/ml leupeptin (Sigma). The mixtures were incubated at 30 °C for 15 min, cooled on ice, and sonicated three times for 10 s using a Branson Sonifier 150 (Branson Ultrasonics, Danbury, CT) at medium setting with intermittent cooling on ice for 30 s. The sonicated cells were centrifuged at 12,000 x g for 20 min at 4 °C, the supernatants recovered, and protein concentrations ascertained with the Bio-Rad protein determination kit. His-tagged p26 was purified from bacterial extracts using 2-ml BD TALON affinity columns as described by the manufacturer (BD Biosciences). The samples were desalted by dialysis against 10 mM NaH2PO4, pH 7.1, and concentrated with Centriprep YM-10 centrifugal devices (Amicon Bioseparations, Billerica, MA).

Bacterial extracts and purified p26 were electrophoresed in 12.5% SDS-polyacrylamide gels followed either by staining with Coomassie Blue or transfer to nitrocellulose. Blots were stained with 2% Ponceau S (Sigma) in 3% trichloroacetic acid to verify protein transfer, washed with TBS-Tween (10 mM Tris-HCl, 140 mM NaCl, 0.1% (v/v) Tween 20, pH 7.4), and incubated for 45 min with shaking in 5% low fat Carnation milk powder in TBS-Tween. The blots were incubated with OmniProbe (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-His antibody diluted in TBS-Tween, followed by goat anti-mouse IgG HRP-conjugated antibody (Jackson ImmunoResearch, Mississauga, ON) diluted in TBS-Tween. Protein detection was conducted with the Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).

Oligomer Formation by Purified p26 —0.5-ml samples of full-length and truncated p26 proteins purified from E. coli were applied to continuous 10-50% (w/v) sucrose gradients and centrifuged at 200,000 x g for 21 h at 4 °C in a Beckman SW41 Ti rotor. Tube bottoms were punctured, 0.8-ml fractions collected, and the A280 of each sample measured with a SPECTRAmax PLUS microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The number of p26 monomers/oligomer was calculated using a p26 molecular mass of 20.8 kDa as determined by GENERUNNER with corrections for the addition and deletion of amino acid residues as necessary. {alpha}-Lactalbumin (14.2 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (BSA) (66 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa) (Sigma) were centrifuged separately and their locations determined by measuring the A280 of gradient fractions.

Chaperone Activity of p26 —A 178 µM stock solution of dimeric citrate synthase (Sigma) was diluted with 40 mM HEPES/KOH buffer at pH 7.5 to a final concentration of 150 nM in 1.0-ml cuvettes and heated at 43 °C with 37.5, 75, 150, 300, and 600 nM purified, bacterially produced p26. p26 molarity in reaction mixtures was based on monomeric molecular masses of p26 variants. Citrate synthase aggregation was monitored by measuring solution turbidity at 360 nm with a SPECTRAmax PLUS spectrophotometer every 2 min for 1 h. BSA (Sigma) and immunoglobulin G (IgG) (Sigma) were used at 600 nM to evaluate nonspecific protection of citrate synthase.

p26 Expression in Transiently Transfected COS-1 Cells—COS-1 cells were maintained at 37 °C under 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 2% penicillin/streptomycin (Invitrogen). Cells in single T75 culture flasks (BD Biosciences) were transfected with mixtures containing 60 µl of Dulbecco's modified Eagle's medium lacking serum and antibiotics, 1 µg of plasmid DNA, and 3 µl of SuperFect (Qiagen). Prior to use, transfection mixtures were incubated at room temperature for 15 min, then placed in T75 flasks containing cells grown to 60% confluence and incubated at 37 °C. The cells were trypsin treated 24 h after transfection, collected by centrifugation at 1,500 x g for 5 min, washed with 1 ml of phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4), centrifuged at 1,500 x g for 5 min, incubated on ice for 20 min in 50 µl of SDS-polyacrylamide gel treatment buffer, and centrifuged at 10,000 x g for 10 min. Supernatant protein concentrations were determined by the Bradford assay (Bio-Rad). Equal volumes of cell-free extract were electrophoresed in 12.5% SDS-polyacrylamide gels and either stained with Coomassie Blue or transferred to nitrocellulose membranes. Immunoprobing of membranes was with OmniProbe as described earlier or with antibody to p26 (39) followed by HRP-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch). p26 Oligomer Formation in COS-1 Cells—Transfected COS-1 cells grown to confluence in T75 culture flasks and collected as described above were incubated on ice for 20 min in 50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, pH 7.8, containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, and 1 µg/ml leupeptin, before centrifugation at 10,000 x g for 10 min. Equal amounts of protein extract from transfected COS-1 cells were applied to individual 10-50% sucrose gradients, prepared by layering 5 ml of 10% (w/v) sucrose on 5 ml of 50% sucrose in 0.1 M Tris/glycine buffer, pH 7.4, in 12-ml tubes, and centrifuging at 200,000 x g for 3 h at 15 °C. The gradients were centrifuged at 200,000 x g for 21 h at 4 °C followed by collection of 0.8-ml fractions. 15 µl from each fraction was electrophoresed in 12.5% SDS-polyacrylamide gels, blotted to nitrocellulose, and probed with either anti-p26 antibody or OmniProbe as described previously.

Immunolocalization of p26 in Transfected COS-1 Cells—60 µl of Dulbecco's modified Eagle's medium without serum or antibiotics, 1 µg of plasmid DNA, and 3 µl of SuperFect were mixed for individual wells of 6-well Falcon culture plates (BD Biosciences), incubated at room temperature for 15 min, placed on cells grown to 60% confluence on coverslips in 6-well plates, and incubated at 37 °C for 24 h. Coverslips were then placed in humidity chambers, rinsed with PBS, and fixed for 20 min at room temperature in 4% paraformaldehyde. The cells were rinsed twice with PBS, permeabilized 5 min in PBS containing 0.2% (v/v) Triton X-100 (PBS-Triton), and incubated at room temperature for 30 min with anti-p26 antibody (39) diluted in PBS-Triton. The cells were washed three times with PBS-Triton then incubated for 20 min at room temperature with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch) diluted in PBS-Triton. After washing three times with PBS-Triton, the cells were incubated with RNase A (Amersham Biosciences) at 5 mg/ml for 10 min and stained with propidium iodide (Sigma) at 0.4 mg/ml for 2 min. Coverslips were rinsed with distilled water and placed on Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Cells expressing His-tagged p26 fusion proteins were also stained with OmniProbe diluted in PBS-Triton, followed by FITC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch). Immunofluorescently stained cells were examined with a Zeiss 410 confocal laser scanning microscope.

Effect of pH on p26 Oligomers—Encysted Artemia embryos (cysts) (INVE Aquaculture, Inc., Ogden, UT) were hydrated in distilled water at 4 °C for 3 h, collected on a Buchner funnel, washed with cold distilled water, and homogenized with a Retsch motorized mortar and pestle (Brinkmann Instruments) in Pipes buffer (100 mM PIPES, 1 mM EGTA, 1mM MgCl2) at either pH 6.5 or 7.0. The homogenates were centrifuged at 16,000 x g for 10 min at 4 °C, and the supernatants were passed through two layers of Miracloth (Calbiochem) before centrifugation at 40,000 x g for 30 min at 4 °C. The upper two-thirds of each supernatant was transferred to a fresh tube and recentrifuged. Either immediately after preparation or after incubation at room temperature for 30 min, supernatants were centrifuged at 200,000 x g for 12 h at 4 °C in 10-50% continuous sucrose gradients at the pH used for homogenization. The gradients were fractionated, and samples were electrophoresed in SDS-polyacrylamide gels, blotted to nitrocellulose, and probed with anti-p26 antibody.

Effect of Heat Shock on p26 Oligomers—10 g of Artemia cysts in 500 ml of distilled H2O was brought to 22 °C, then heated to 50 °C over 1 h with vigorous aeration in a Programmable/Digital Immersion Circulator (VWR, Mississauga, ON). Heat-shocked and control cysts, the latter incubated on ice for 30 min, were homogenized separately by hand for 2 min in a chilled mortar and pestle in 35 ml of cold HPC (1 mM CaCl2, 0.05 mM PIPES, 6.4% hexylene glycol, pH 7.0), followed by one passage in a Dounce homogenizer, filtration through Miracloth, and centrifugation at 2,000 x g for 10 min at 4 °C to pellet nuclei. Supernatants were centrifuged at 40,000 x g for 30 min at 4 °C, and the upper two-thirds was placed in a fresh tube and centrifuged. Supernatants were centrifuged in 10-50% continuous sucrose gradients, fractionated, and analyzed on Western blots after SDS-PAGE.

The nuclei-containing pellets from above were rinsed twice with 40 ml of HPC, resuspended in 17.5 ml of the same buffer, applied to 25-ml cushions of 75% (v/v) Percoll (Sigma) in 150 mM NaCl, 14 mM MgCl2, 16 mM Tris, pH 7.0, and centrifuged at 16,000 x g for 30 min at 4 °C in a Beckman JS-13.1 swinging bucket rotor. Cloudy layers were transferred to fresh tubes, brought to 15 ml with HPC, placed on a 25-ml Percoll cushion, and centrifuged as above. Nuclei were recovered, mixed with an equal volume of HPC, centrifuged at 16,000 x g for 30 min at 4 °C, and the nuclei-containing pellets were suspended in 1.0 ml of HPC. Samples were stained with 0.4% DAPI (Molecular Probes, Eugene, OR) and examined microscopically. Nuclei from control and heat-shocked embryos were lysed in treatment buffer, electrophoresed in 12.5% SDS-polyacrylamide gels, blotted to nitrocellulose, and probed with anti-p26 antibody to determine whether p26 had moved into nuclei. For sucrose density gradient centrifugation, extracts were prepared by suspending purified nuclei in 1.0 ml of ice-cold extraction buffer (20 mM HEPES/KOH, 2.5 mM MgCl2, 0.42 M NaCl, 25% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, pH 7.9) containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, and 1 µg/ml leupeptin and sonicating three times for 10 s using a Branson Sonifier 150 at medium setting with intermittent cooling on ice for 30 s. The sonicated samples were incubated on ice for 30 min and then centrifuged at 25,000 x g for 30 min at 4 °C. Nuclear extracts were centrifuged in 10-50% continuous sucrose gradients at 200,000 x g for 12 h at 4 °C. The gradients were fractionated, and samples were electrophoresed in SDS-polyacrylamide gels, blotted to nitrocellulose, and probed with antibody to localize p26.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of p26 cDNAs—Full-length and truncated p26 cDNAs cloned in the prokaryotic expression vector pRSET.C included p26-full-His, the full-length polypeptide; p26-N{Delta}36-His, residues 1-36 deleted; p26-N{Delta}60-His, residues 1-60 removed; p26-C{Delta}40-His, final 40 residues eliminated; p26-C{Delta}10-His, lacked the last 10 residues; p26-{alpha}-His, missing residues 1-60 and 153-192, thereby consisting of the {alpha}-crystallin domain (Fig. 1). All p26 cDNAs generated by site-directed mutagenesis contained, in addition to the p26 sequence, an amino-terminal peptide of 13 residues encoded by p26-3-6-3 and the His tag. The presence of the His tag is indicated by "-His" in the name of the p26 cDNA or protein. The p26 cDNAs recovered from the storage vector pCRII (37) were cloned in pSecCMV for expression in COS-1 cells. p26-full, p26-N{Delta}60, and p26-{alpha} were also prepared in pcDNA4/His.A for expression as His-tagged fusion proteins. All cloned p26 cDNA fragments were sized by electrophoresis in agarose after restriction digestion of recombinant plasmids, and PCR fidelity was confirmed by sequencing all cloned inserts (not shown).



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  		FIG. 1.
Schematic representation and sequence of p26 cDNAs. a, p26 cDNAs generated by site-directed mutagenesis and PCR were cloned in the prokaryotic expression vector pRSET.C and the eukaryotic expression vectors pSecCMV and pcDNA4/His.A. The primers used for site-directed mutagenesis are listed in Table I. The box labeled 6xHis is the His tag-encoding sequence present in the vectors pRSET.C and pcDNA4/His.A. b, amino acid sequence of p26 (accession number AF031367 [GenBank] ). The amino acid residues removed to generate p26-N{Delta}60 and p26-C{Delta}40 are shaded, and those for p26-N{Delta}36 and p26-C{Delta}10 are shaded and bold.

 
p26 Oligomer Formation and Chaperone Activity—Isopropyl-1-thio-{beta}-D-galactopyranoside induction of transformed E. coli yielded polypeptides on Western blots of the appropriate size that reacted strongly with OmniProbe, although bands corresponding to p26 were not readily visible in Coomassie Blue-stained gels (Fig. 2, a and b). With the exception of p26-N{Delta}60-His and p26-{alpha}-His, all of the polypeptides were recognized by antibody to p26 (not shown). Subsequent to purification on TALON affinity columns, single bands of the expected size appeared on Coomassie Blue-stained gels (Fig. 2c). These polypeptides reacted strongly with OmniProbe (Fig. 2d), indicating that they were His-tagged p26, although purified p26-N{Delta}60-His and p26-{alpha}-His were again not recognized by anti-p26 antibody. Protein bands were not detected on blots containing extract from E. coli transformed with vector lacking p26 cDNA (Fig. 2, a and b, lane 7).



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  		FIG. 2.
Purification of p26 synthesized in E. coli. Cell-free extracts from transformed E. coli induced with isopropyl-1-thio-{beta}-D-galactopyranoside were electrophoresed in SDS-polyacrylamide gels and either stained with Coomassie Blue (a) or blotted to nitrocellulose and reacted with OmniProbe (b). Proteins purified by affinity chromatography were electrophoresed in SDS-polyacrylamide gels and either stained with Coomassie Blue (c) or blotted to nitrocellulose and reacted with OmniProbe (d). The lanes contained p26-N{Delta}36-His (lane 1), p26-N{Delta}60-His (lane 2), p26-{alpha}-His (lane 3), p26-C{Delta}40-His (lane 4), p26-C{Delta}10-His (lane 5), p26-full-His (lane 6), and vector lacking p26 cDNA (lane 7). All lanes received 10 µl of sample. M, molecular mass markers of 97, 66, 45, 31, 21, and 14 kDa.

 
The molecular mass of oligomers assembled from purified p26-full-His synthesized in E. coli ranged from 26 to 600 kDa, with the peak at about 150 kDa, or 6 monomers/oligomer (Fig. 3 and Table II). The oligomers assembled with full-length p26 were larger than those obtained with truncated proteins, although oligomers of p26-C{Delta}10-His were similar in mass. At the other extreme, p26-{alpha}-His migrated predominantly as monomers, and p26-N{Delta}60-His existed as monomers and dimers, although larger complexes occurred. Oligomers assembled from other p26 variants were intermediate in mass to those just described.



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  		FIG. 3.
Oligomer formation by purified p26. Bacterially produced p26 purified to apparent homogeneity by affinity chromatography was centrifuged at 200,000 x g for 21 h at 4 °C on 10-50% sucrose gradients. The gradients were fractionated, and the A280 of each fraction was plotted against fraction number. The top of each gradient is on the right. The molecular mass markers ({alpha}-lactalbumin, 14.2 kDa; carbonic anhydrase, 29 kDa; BSA, 66 kDa; alcohol dehydrogenase, 150 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa) are indicated by numbered arrows.

 


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  		TABLE II
Oligomerization of p26 produced in E. coli

The molecular mass of p26 oligomers produced by transformed E. coli was determined by reading the A280 of samples obtained by fraction-action of sucrose density gradients. Monomer mass refers to the molecular mass of p26 polypeptides. Oligomer mass range represents the smallest to largest oligomers observed, and oligomer mass peak refers to the mass of the most prevalent oligomer. Monomer number peak refers to the number of subunits in the most prevalent oligomer.

 
Purified p26-full-His possessed the greatest chaperone activity and p26-{alpha}-His the least, as determined by heat-induced denaturation of citrate synthase, although all variants provided some protection (Fig. 4). At a chaperone:target molar ratio of 4:1 (p26 monomer:citrate synthase dimer), or 1:1.5 if the peak oligomer size of p26-full-His is used for comparison, citrate synthase protection was almost complete after 1 h at 43 °C (Fig. 4a). At a p26-full-His:citrate synthase 1:4 molar ratio or oligomer:dimer 1:24 ratio, purified p26 reduced the heat-induced turbidity increase by ~25% (Fig. 4e). In contrast, p26-{alpha}-His was almost devoid of chaperone activity even at molar ratios of 4:1 (Fig. 4). p26-N{Delta}60-His was marginally better than p26-{alpha}-His as a chaperone, followed by p26-N{Delta}36-His and p26-C{Delta}40-His, which were similar to one another. p26-C{Delta}10-His approached p26-full-His in chaperone potency especially at high concentrations. BSA and IgG at 600 nM provided almost no protection upon heating of citrate synthase (Fig. 4f).



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  		FIG. 4.
p26 chaperone activity. Bacterially produced p26 purified to apparent homogeneity was heated at 43 °C for 1 h with 150 nM citrate synthase, and solution turbidity was measured at A360. p26 was at final concentrations of: 600 nM (a); 300 nM (b), 150 nM (c), 75 nM (d), or 37.5 nM (e). The curves are: 1, no p26; 2, p26-{alpha}-His; 3, p26-N{Delta}60-His; 4, p26-N{Delta}36-His; 5, p26-C{Delta}40-His; 6, p26-C{Delta}10-His; 7, p26-full-His, and they occupy the same relative positions in a-e; f contains 150 nM citrate synthase incubated in the absence of other proteins (1) and with either 600 nM BSA (2) or 600 nM IgG (3).

 
Synthesis and Oligomerization of p26 in Transfected Mammalian Cells—Except for p26-N{Delta}60 and p26-{alpha}, which reacted with OmniProbe, protein extracts from COS-1 cells transfected with p26 cDNA-containing expression vectors yielded polypeptides of the expected size on Western blots with anti-p26 antibody (Fig. 5). Neither primary antibody gave protein bands on blots with extracts from COS-1 cells transfected with vectors lacking p26 cDNA (not shown). Full-length p26 synthesized in COS-1 cells produced oligomers as large as 512 kDa and composed of up to 21 monomers, with monomer number essentially the same in the presence and absence of the His tag (Fig. 6, a and b). Oligomers assembled with p26-C{Delta}10 were only slightly smaller than those produced from full-length p26 (Fig. 6c), but removal of the complete carboxyl-terminal extension gave oligomers of a narrower size range and smaller mass (Fig. 6d). Oligomer mass became progressively smaller upon sequential removal of the amino terminus (Fig. 6, e and f) with polypeptides encoded by p26-N{Delta}60-His present as monomers and dimers, as was true for p26-{alpha}-His (Fig. 6g), although the latter was more enriched in monomers. The properties of p26 oligomers produced in COS-1 cells are summarized in Table III.



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  		FIG. 5.
p26 synthesis in transfected COS-1 cells. Extracts prepared from COS-1 cells transfected with the pSecCMV eukaryotic expression vector containing p26 cDNAs were electrophoresed in SDS-polyacrylamide gels and either stained with Coomassie Blue (a) or transferred to nitrocellulose and stained with anti-p26 antibody followed by HRP-conjugated goat anti-rabbit IgG antibody (b). Each lane received 10 µl of extract from cells transfected with p26-N{Delta}36 (lane 1), p26-N{Delta}60 (lane 2), p26-{alpha} (lane 3), p26-C{Delta}40 (lane 4), p26-C{Delta}10 (lane 5), or p26-full (lane 6). M, molecular mass markers of 97, 66, 45, 31, 21, and 14 kDa. Western blots containing extracts from COS-1 cells transfected with the pcDNA4/His.A expression vector containing p26 cDNAs and prepared as just described were stained with either anti-p26 antibody followed by HRP-conjugated goat anti-rabbit IgG antibody (c) or OmniProbe followed by HRP-conjugated goat anti-mouse IgG (d). Each lane in c and d received 10 µl of extract from cells transfected with p26-full-His (lane 1), p26-N{Delta}60-His (lane 2), or p26-{alpha}-His (lane 3).

 



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  		FIG. 6.
p26 oligomer formation in transfected COS-1 cells. Extracts prepared from COS-1 cells transiently transfected with p26-containing vectors were centrifuged in 10-50% sucrose gradients, and 15-µl samples from each fraction were electrophoresed in 12.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with either anti-p26 antibody followed by HRP-conjugated goat anti-rabbit IgG (a, c, d, and e) or OmniProbe followed by HRP-conjugated goat anti-mouse IgG (b, f, and g). The gradients contained p26-full (a), p26-full-His (b), p26-C{Delta}10 (c), p26-C{Delta}40 (d), p26-N{Delta}36 (e), p26-N{Delta}60-His (f), and p26-{alpha}-His (g). The top of each gradient is on the right, and fractions are numbered across the top. The positions of molecular mass markers ({alpha}-lactalbumin, 14.2 kDa; carbonic anhydrase, 29 kDa; BSA, 66 kDa; alcohol dehydrogenase, 150 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa) are indicated by numbered arrows.

 


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  		TABLE III
Oligomerization of p26 synthesized in transfected COS-1 cells

The molecular mass of p26 oligomers was determined by immunoprobing samples electrophoresed in SDS-polyacrylamide gels and transferred to Western blots after sucrose gradient centrifugation. Monomer mass refers to the molecular mass of p26 polypeptides. Oligomer mass range represents the smallest to largest oligomers observed, and oligomer mass peak refers to the mass of the most prevalent oligomer. Monomer number peak refers to the number of subunits in the most prevalent oligomer.

 
Truncated p26 Localizes to COS-1 Nuclei—To monitor p26 synthesis transiently transfected COS-1 cells were stained with anti-p26 antibody, OmniProbe, and propidium iodide, revealing that polypeptides encoded by p26-full and p26-full-His (Fig. 7, a and b) and the carboxyl-terminal truncations, p26-C{Delta}40 (Fig. 7c) and p26-C{Delta}10 (Fig. 7d), localized exclusively to the cytoplasm. In contrast, upon amino-terminal modification p26 occurred in cytoplasm and nuclei, the latter shown by yellow staining. p26-N{Delta}36 encoded polypeptides were in the nuclei of only some cells (Fig. 7, e and f), but polypeptides p26-N{Delta}60/p26-N{Delta}60-His lacking the amino terminus (Fig. 7, g and h), or p26-{alpha}/p26-{alpha}-His composed of the {alpha}-crystallin domain (Fig. 7, i and j), resided in the nuclei of all transfected cells, indicating that the His tag had no effect on p26 localization. The results suggested that disassembly of oligomers is responsible for p26 movement into nuclei of COS-1 cells and by extrapolation, the nuclei of encysted Artemia embryos.



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  		FIG. 7.
p26 localization in transfected COS-1 cells. COS-1 cells transfected with the p26 cDNA-containing vector pSecCMV were incubated with antibody to p26 followed by FITC-conjugated goat anti-rabbit IgG antibody (green) (a, c, d, e, and f). Cells transfected with the p26 cDNA-containing vector pcDNA4/His.A were exposed to anti-p26 antibody followed by FITC-conjugated goat anti-rabbit IgG (green) (g and i) or to OmniProbe followed by FITC-conjugated goat anti-mouse IgG (green) (b, h, and j). Nuclei were stained with propidium iodide (red), and samples were examined by confocal microscopy. a, p26-full; b, p26-full-His; c, p26-C{Delta}40; d, p26-C{Delta}10; e and f, p26-N{Delta}36; g, p26-N{Delta}60; h, p26-N{Delta}60-His; i, p26-{alpha}; j, p26-{alpha}-His. The scale bar represents 50 µm, and all figures are the same magnification.

 
Oligomer Size of p26 from Artemia Embryos Is Unaffected by pH and Heat Shock—p26 moves into the nuclei of Artemia embryos upon exposure to reduced pH in vitro and upon heat shock in vivo by an unknown mechanism that, as just suggested by the previous results, entails oligomer mass reduction. However, the p26 oligomers obtained from Artemia cysts homogenized at either pH 6.5 or 7.0 were identical in molecular mass, whether or not they were incubated 30 min at room temperature before gradient centrifugation (Fig. 8, a-d). p26 migrated into the nuclei of heat-shocked Artemia embryos under the conditions used in this work (Fig. 9, a and b), but the size of p26 oligomers in extracts from cysts (Fig. 9, c and d) and nuclei (Fig. 9, e and f) remained constant. The p26 in nuclear extracts tended to smear upon electrophoresis and blotting, probably because of the presence of DNA.



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  		FIG. 8.
p26 oligomer size is unaffected by pH. Protein extracts obtained from Artemia cysts prepared at either pH 7.0 (a and c) or pH 6.5 (b and d) were centrifuged in 10-50% continuous sucrose gradients either immediately after preparation (a and b) or after incubation at room temperature for 30 min (c and d). 15-µl fractions from each gradient were electrophoresed in 12.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with anti-p26 antibody followed by HRP-conjugated goat anti-rabbit IgG. The top of each gradient is on the right, and fractions are numbered across the top. The positions of molecular mass markers ({alpha}-lactalbumin, 14.2 kDa; carbonic anhydrase, 29 kDa; BSA, 66 kDa; alcohol dehydrogenase, 150 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa) are indicated by numbered arrows.

 



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  		FIG. 9.
p26 oligomer size is unaffected by heat shock. Extracts prepared from heat-shocked Artemia cysts and their nuclei were electrophoresed in SDS-polyacrylamide gels and either stained with Coomassie Blue (a) or blotted to nitrocellulose and stained with anti-p26 antibody (b). The lanes contained: total cyst extract, heat shocked (lane 1); total cyst extract, control (lane 2); nuclear extract, heat shocked (lane 3); nuclear extract, control (lane 4). Total extract from heated (c) and control (d) cysts and nuclear extracts from heated (e) and control (f) cysts were centrifuged in continuous 10-50% sucrose gradients, and 15-µl fractions from each gradient were electrophoresed in SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with anti-p26 antibody followed by HRP-conjugated goat anti-rabbit IgG. The top of each gradient is on the right, and fractions are numbered across the top. The positions of molecular mass markers ({alpha}-lactalbumin, 14.2 kDa; carbonic anhydrase, 29 kDa; BSA, 66 kDa; alcohol dehydrogenase, 150 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa) are indicated by numbered arrows.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Full-length p26 produced in either bacteria or mammalian cells yielded oligomers that were somewhat smaller than those from encysted Artemia embryos but represented effective oligomerization nonetheless. In contrast, the {alpha}-crystallin domain existed mainly as monomers and dimers, as was true for p26 lacking the entire amino terminus. These data indicate a role for the amino terminus in formation of dimers and thus higher oligomers, a conclusion strengthened by the comparatively greater oligomerization of p26 lacking 36 rather than 60 residues. The {alpha}-crystallin domain and amino-terminal truncated p26 variants were poor chaperones in relation to full-length p26, with chaperone activity decreasing as truncation increased. Reduced chaperone activity and oligomerization were likely caused by the loss of residues involved in oligomer formation and interaction with substrates. As one example, the p26 motif 17WSDPF21 corresponds to 15SWEPF19 in Chinese hamster Hsp27, a sequence important for oligomer formation and chaperoning (45). p26 structural organization and function may also depend on other hydrophobic elements of the amino-terminal region with the deleted residues 1-36 and 1-60 possessing 72.2 and 61.7% hydrophobicity, respectively, both significantly higher than the 51% hydrophobicity of the full-length protein.

Compared with p26, yeast Hsp26 lacking amino-terminal sequences formed dimers devoid of chaperone activity even though dissociation of oligomers into dimers is a functional prerequisite (8). Human {alpha}B-crystallin missing amino acid residues 1-56 produced dimers with significant chaperone activity (46), whereas removing either 50 or 56 residues from the amino terminus of {alpha}A-crystallin decreased oligomer mass from 550 to 60 kDa, the latter composed of tetramers and dimers (47). The results indicate that sHsp dimerization occurs independently of the amino-terminal domain, but formation of larger oligomers requires this region, conclusions in partial agreement with the findings for p26. Hsp16.5 from M. jannaschii (19) and Hsp16.9 from T. aestivum (18) assemble well defined (monodisperse) oligomers, and for Hsp16.5 the amino terminus facilitates oligomerization but does not necessarily determine the final architectural structure (48). Deletion of 42 amino-terminal residues from rice Hsp16.9 decreased chaperone activity but not oligomer size (49), and in the bacterium Bradyrhizobium japonicum, where the amino terminus is required for oligomer assembly, at least a portion of the region drives dimer formation (17), as appears to be true for p26.

Removal of 10 carboxyl-terminal residues had little effect on p26; however, deleting the entire carboxyl terminus, including the conserved motif I/V-X-I/V (as VPI) (17), reduced oligomerization and chaperoning. The carboxyl terminus of p26 was not required for dimer formation, but the region contributed to oligomer assembly, although less so than the amino terminus. Deletion of the entire p26 carboxyl-terminal extension reduced protection of citrate synthase upon heating, indicating a role in chaperoning which may depend upon oligomerization, recognition of substrate proteins, chaperone/substrate solubility, or a combination of these. Precipitation of truncated p26 was not observed even with complete removal of the carboxyl terminus, which contains 20 polar and 4 charged amino acids in the final 30 residues. In contrast, loss of the last 16 amino acids from Caenorhabditis elegans Hsp16-2 was without effect on oligomer size and chaperone activity, but the modified protein precipitated upon freeze/thawing (50), suggesting reduced solubility.

The carboxyl terminus promotes sHsp/substrate solubility and chaperoning to varying degrees (17, 50-53). Eliminating the last 18 amino acid residues from mouse Hsp25, which excludes the conserved I/V-X-I/V motif (17), had little effect on oligomerization and chaperoning of citrate synthase at 43 °C, but protection of {alpha}-lactalbumin against dithiothreitol-induced denaturation was lost (52). Deletion of 10 carboxyl-terminal residues has minimal impact on {alpha}A-crystallin and sometimes even enhanced substrate protection, albeit modestly (14, 47), but this result was never obtained with p26. In contrast, removal of 11 or more C-terminal residues from {alpha}A-crystallin, including the I/V-X-I/V motif, drastically reduced oligomer size and chaperoning, with Arg-163 particularly important. Additionally, the carboxyl terminus of the B. japonicum sHsp, and especially the conserved motif I/V-X-I/V, plays a role in oligomer assembly (17), as is true for Pfu-sHsp from the hyper-thermophilic microorganism Pyrococcus furiosus (54), all results in agreement with those obtained with p26.

The role of sHsps in nuclei has received limited attention. {alpha}B-crystallin and Hsp27 associate with nuclear speckles and nucleoli of various human cell lines in nonstress conditions and may exert regulatory roles in these locations (55, 56). Human Hsp27 translocates into nuclei of transfected A549 cells encountering stress, although protection occurs independently of nuclear localization (57). p26 migrates into nuclei early in oviparous development (38), during physiological stress and upon exposure to acidic conditions in vitro (42-44). A nuclear localization signal as occurs in tomato Hsp16.1-CIII (58) is not apparent, but residues 36-45 of p26 include 6 arginines (39), a potential nuclear localization signal.

In this study, full-length and carboxyl truncated p26 resided exclusively in transfected COS-1 cell cytoplasm. In contrast, complete removal of the amino-terminal domain, including the putative nuclear localization signal, resulted in nuclear translocation. One interpretation of this finding is that decreased oligomer disassembly permits p26 movement through nuclear pores by simple diffusion, and nuclear translocation of rat Hsp20 and Hsp25 upon heat shock, where they may play protective roles, is accompanied by stress-induced decrease in oligomer mass, perhaps to dimers for Hsp20 (59). However, p26, reduced in oligomer size because of carboxyl-terminal truncation, remained in the cytoplasm, suggesting that simple diffusion is not occurring. In support of this, modification of a single p26 residue by site-directed mutagenesis had little effect on oligomerization but led to nuclear localization.2 Additionally, p26 oligomers in heat-stressed and control cysts, including those from nuclei, were similar, and their mass was maintained in reduced pH, a condition that promotes p26 translocation into nuclei in vivo and in vitro. The most direct conclusion is that p26 migration into cyst nuclei depends on a mechanism other than oligomer mass reduction, although the transient formation of monomers or dimers followed by reassembly in the nucleus is possible. Clearly, however, movement of p26 into the nuclei occurs independently of the arginine-enriched amino-terminal sequence.

To conclude, the amino-terminal domain of p26, a sHsp occurring in embryos that exhibit extreme stress resistance, promotes {alpha}-crystallin dimerization and in concert with the carboxyl-terminal extension enhances protein oligomerization. As measured by heat-induced denaturation of citrate synthase, the {alpha}-crystallin domain of p26 lacks effective chaperone activity, depending on amino- and carboxyl-terminal regions for full function. The nuclear translocation of p26 in COS-1 cells occurs independently of oligomer size and a putative nuclear localization signal in the amino-terminal region, suggesting a complex mechanism for movement into the nuclei of encysted Artemia embryos. Additional work to support these conclusions and to define domain-specific p26 amino acid residues with structural/functional implications is under way.


   FOOTNOTES
 
* This work was supported in part by a Natural Sciences and Engineering Research Council of Canada discovery grant and a regional partnership plan grant including support from the Nova Scotia Health Research Foundation, the Canadian Institutes of Health Research, and the Heart and Stroke Foundation of Nova Scotia (to T. H. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by a Nova Scotia Health Research Foundation student research award. Back

§ To whom correspondence should be addressed: Dept. of Biology, Dalhousie University, 1355 Oxford St., Halifax, NS B3H 4J1, Canada. Tel.: 902-494-6525; Fax: 902-494-3736; E-mail: tmacrae{at}dal.ca.

1 The abbreviations used are: sHsp, small heat shock protein; BSA, bovine serum albumin; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid. Back

2 Y. Sun and T. H. MacRae, manuscript in preparation. Back



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