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• W2022038675 abstract "Plants synthesize several classes of small heat shock proteins ranging in size from 15 to 30 kDa. Two conserved classes, designated class I and class II, are localized to the cytosol. Recombinant HSP18.1 and HSP17.7, representing class I and class II proteins from pea, respectively, were expressed in Escherichia coli and purified. Non-denaturing polyacrylamide gel electrophoresis and electron microscopy demonstrated that the purified proteins formed discretely sized, high molecular weight complexes. Sedimentation equilibrium analytical ultracentrifugation revealed that the HSP18.1 and HSP17.7 complexes were composed of approximately 12 subunits. Both proteins were able to enhance the refolding of chemically denatured citrate synthase and lactate dehydrogenase at stoichiometric levels in an ATP-independent manner. Furthermore, HSP18.1 and HSP17.7 prevented aggregation of citrate synthase at 45°C and irreversible inactivation of citrate synthase at 38°C. HSP18.1 also suppressed aggregation of lactate dehydrogenase at 55°C. These findings demonstrate that HSP18.1 and HSP17.7 can function as molecular chaperones in vitro. Plants synthesize several classes of small heat shock proteins ranging in size from 15 to 30 kDa. Two conserved classes, designated class I and class II, are localized to the cytosol. Recombinant HSP18.1 and HSP17.7, representing class I and class II proteins from pea, respectively, were expressed in Escherichia coli and purified. Non-denaturing polyacrylamide gel electrophoresis and electron microscopy demonstrated that the purified proteins formed discretely sized, high molecular weight complexes. Sedimentation equilibrium analytical ultracentrifugation revealed that the HSP18.1 and HSP17.7 complexes were composed of approximately 12 subunits. Both proteins were able to enhance the refolding of chemically denatured citrate synthase and lactate dehydrogenase at stoichiometric levels in an ATP-independent manner. Furthermore, HSP18.1 and HSP17.7 prevented aggregation of citrate synthase at 45°C and irreversible inactivation of citrate synthase at 38°C. HSP18.1 also suppressed aggregation of lactate dehydrogenase at 55°C. These findings demonstrate that HSP18.1 and HSP17.7 can function as molecular chaperones in vitro. Plants synthesize numerous small heat shock proteins (smHSPs)1( 1The abbreviations used are: smHSPsmall heat shock proteinCScitrate synthaseLDHlactate dehydrogenasePAGEpolyacrylamide gel electrophoresis. ) ranging in size from 15 to 30 kDa that are related to the evolutionarily conserved family of smHSPs and α-crystallins of the vertebrate eye lens (1Vierling E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 579-620Crossref Scopus (1374) Google Scholar, 2Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 70: 2360-2364Crossref Scopus (671) Google Scholar). In contrast to mammalian smHSPs, plant smHSPs constitute the most abundant and diverse group of proteins synthesized in response to heat stress. For example, more than 25 individual smHSP polypeptides can be observed by two-dimensional PAGE from a variety of heat-stressed plants (3Mansfield M.A. Key J.L. Plant Physiol. 1987; 84: 1007-1017Crossref PubMed Google Scholar). Only one species of smHSP has been identified in human, mouse, and yeast, and each is typically present in the cytosol (4Arrigo A.-P. Landry J. Morimoto R. Tissieres A. Georgeopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, NY1994: 335-373Google Scholar). At least four major classes of plant smHSPs have been identified based on sequence alignments and immunological cross-reactivity; two are found in the cytosol, one is localized to the chloroplast, and another is localized to the endoplasmic reticulum (1Vierling E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 579-620Crossref Scopus (1374) Google Scholar). Recently, a plant smHSP localized to mitochondria has also been identified (5Lenne C. Douce R. Plant Physiol. 1994; 105: 1255-1261Crossref PubMed Scopus (55) Google Scholar). small heat shock protein citrate synthase lactate dehydrogenase polyacrylamide gel electrophoresis. Little is known about the cellular function of smHSPs, although evidence from mammalian and Drosophila systems supports the theory that smHSPs play a role in thermotolerance (6Rollet E. Lavoie J.N. Landry J. Tanguay R.M. Biochem. Biophys. Res. Commun. 1992; 185: 116-120Crossref PubMed Scopus (73) Google Scholar, 7Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar, 8Berger E.M. Woodward M.P. Exp. Cell Res. 1983; 147: 437-442Crossref PubMed Scopus (88) Google Scholar). Recently, it has been demonstrated that mammalian smHSPs and the related α-crystallins possess molecular chaperone activity in vitro(9Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1726) Google Scholar, 10Merck K.B. Groenen P.J.T.A. Voorter C.E.M. de Haard-Hoekman W.A. Horwitz J. Bloemendal H. de Jong W.W. J. Biol. Chem. 1993; 268: 1046-1052Abstract Full Text PDF PubMed Google Scholar, 11Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar). In contrast to the ATP-dependent molecular chaperones of the HSP60 and HSP70 classes (12Hendrick J.P. Hartl F.-U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1457) Google Scholar), the smHSPs were able to refold chemically denatured proteins in an ATP-independent manner (9Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1726) Google Scholar, 10Merck K.B. Groenen P.J.T.A. Voorter C.E.M. de Haard-Hoekman W.A. Horwitz J. Bloemendal H. de Jong W.W. J. Biol. Chem. 1993; 268: 1046-1052Abstract Full Text PDF PubMed Google Scholar, 11Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar). More recent data have suggested that mammalian smHSPs may regulate actin filament dynamics (7Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar, 13Miron T. Vancompernolle K. Vanderkerckhove J. Wilchek M. Geiger B. Cell Biol. 1991; 114: 255-261Crossref PubMed Scopus (385) Google Scholar, 14Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar). Small HSPs from diverse organisms typically form multimeric complexes ranging in size from 200 to 800 kDa (1Vierling E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 579-620Crossref Scopus (1374) Google Scholar, 4Arrigo A.-P. Landry J. Morimoto R. Tissieres A. Georgeopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, NY1994: 335-373Google Scholar). The complexes from plant sources are small and uniform, from 200 to 240 kDa (5Lenne C. Douce R. Plant Physiol. 1994; 105: 1255-1261Crossref PubMed Scopus (55) Google Scholar, 16Helm K.W. LaFayette P.R. Nagao R.T. Key J.L. Vierling E. Mol. Cell. Biol. 1993; 13: 238-247Crossref PubMed Scopus (77) Google Scholar, 17Chen Q. Osteryoung K. Vierling E. J. Biol. Chem. 1993; 269: 13216-13223Abstract Full Text PDF Google Scholar). Mammalian smHSPs generally form larger complexes from 500 to 800 kDa (18Arrigo A.-P. Welch W.J. J. Biol. Chem. 1987; 262: 15359-15369Abstract Full Text PDF PubMed Google Scholar, 19Behlke J. Lutsch G. Gaestel M. Bielka H. FEBS Lett. 1991; 288: 119-122Crossref PubMed Scopus (59) Google Scholar), but their oligomeric structure appears to be dynamic in response to phosphorylation (20Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar, 21Benndorf R. Hayeβ K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). Consequently, the actual size of mammalian smHSP complexes has been a matter of controversy. Phosphorylation of plant smHSPs, however, has not been observed (22Nover L. Scharf K.-D. Eur. J. Biochem. 1984; 139: 303-313Crossref PubMed Scopus (100) Google Scholar). Based on sequence alignments, all plants synthesize two distinct classes of cytosolic smHSPs, designated class I and class II (1Vierling E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 579-620Crossref Scopus (1374) Google Scholar). HSP18.1, a class I protein, and HSP17.7, a class II protein, both from Pisum sativum (pea), share 60% amino acid similarity and 38% identity (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar). These two proteins, with derived molecular masses of 18,086 and 17,732 Da for HSP18.1 and HSP17.7, respectively, are normally absent from vegetative tissues in plants unless induced by heat at temperatures above 30°C (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar). Maximum induction of HSP18.1 occurs at 38°C, during which class I smHSP levels can reach up to 1% of the total protein in roots and somewhat lower levels in leaves (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar). Following heat stress, accumulated HSP18.1 remains stable with a half-life of 38 h (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar) and may therefore function during both heat stress and recovery. HSP18.1 and HSP17.7 share marginal overall sequence homology with mammalian smHSPs. For example, HSP18.1 shares only 25% identity with human HSP27 at the amino acid level (23Plesovsky-Vig N. Vig J. Brambl R. J. Mol. Evol. 1992; 35: 537-545Crossref PubMed Scopus (59) Google Scholar). However, like all smHSPs and α-crystallins, the regions of greatest homology occur in the C-terminal halves of the proteins (23Plesovsky-Vig N. Vig J. Brambl R. J. Mol. Evol. 1992; 35: 537-545Crossref PubMed Scopus (59) Google Scholar). HSP18.1 and HSP17.7 expression is not strictly limited to heat stress conditions. Both proteins have been identified in developing embryos at levels roughly equivalent to those observed in moderately heat-stressed tissues (24DeRocher A.E. Vierling E. Plant J. 1994; 5: 93-102Crossref Scopus (106) Google Scholar). Cytosolic smHSPs have also been shown to be expressed during pollen development (25Bouchard R.A. Genome. 1990; 33: 68-79Crossref PubMed Scopus (48) Google Scholar, 26Dietrich P.S. Bouchard R.A. Casey E.S. Sinibaldi R.M. Plant Physiol. 1991; 96: 1268-1276Crossref PubMed Scopus (40) Google Scholar), and corresponding mRNAs have been identified in ripening fruit (27Fray R.G. Lycett G.W. Grierson D. Nucleic Acids Res. 1990; 18: 7148Crossref PubMed Scopus (32) Google Scholar). How these other patterns of smHSP expression are related to their presence during and after heat stress is not understood, but developmentally regulated patterns of smHSP expression have also been observed in Drosophila and mammalian cells (reviewed in Ref. 4Arrigo A.-P. Landry J. Morimoto R. Tissieres A. Georgeopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, NY1994: 335-373Google Scholar). Because of the abundance and diversity of smHSPs in plants, we have been particularly interested in understanding and comparing the structural and functional properties of plant smHSPs. Here, we have characterized recombinant HSP18.1, a class I smHSP, and HSP17.7, a class II smHSP, with regard to oligomeric structure and in vitro molecular chaperone activity. Pig heart citrate synthase, bovine muscle lactate dehydrogenase, bovine liver catalase, bovine IgG, Tris, and HEPES were obtained from Sigma. Ultrapure (NH4)2SO4 and ultrapure urea were obtained from Baker. Ultrapure guanidine hydrochloride was obtained from Life Technologies, Inc. The Pshsp18.1 and Pshsp17.7 cDNAs (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar, 28Lauzon L. Helm K.W. Vierling E. Nucleic Acids Res. 1990; 18: 4274Crossref PubMed Scopus (29) Google Scholar), which encode pea HSP18.1 and HSP17.7, respectively, were subcloned into the expression vector pJC20 (29Clos J. Brandau S. Protein Expression Purif. 1994; 5: 133-137Crossref PubMed Scopus (148) Google Scholar) as follows. The EcoRI fragment of Pshsp18.1 was end-filled with Klenow fragment and inserted by blunt-end ligation into the BamHI site of pJC20 that had also been end-filled. The SmaI/ApaI fragment of Pshsp17.7 was inserted into the similarly prepared pJC20. To eliminate N-terminal fusion proteins encoded by these pJC20 derivatives, polymerase chain reaction mutagenesis was used to introduce NdeI sites at the translation start sites of the Pshsp18.1 and Pshsp17.7 cDNAs. The PCR products were subcloned into vector pCR using the TA cloning system (Invitrogen). The NdeI/NcoI fragment from the modified Pshsp18.1 cDNA and the NdeI fragment from the modified Pshsp17.7 cDNA were exchanged for the corresponding wild-type fragments in the pJC20 derivatives to yield pAZ316 and pAZ317, respectively. The engineered mutations were confirmed by dideoxy sequencing (30Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar) of the regions derived from the polymerase chain reaction. Escherichia coli BL21(DE3) cells were transformed with pAZ316 or pAZ317 for T7 RNA polymerase-directed expression of the target proteins (31Studier F.W. Moffat B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4772) Google Scholar). Exponentially growing cells were induced with 1 mM isopropyl β-D-thiogalactopyranoside for 6 h and then harvested. Cells were washed with 50 mM Tris-HCl, 1 mM EDTA, pH 7.5 (buffer A), broken by sonic lysis, and centrifuged at 17,500 × g for 30 min to yield soluble crude extracts. Crude extracts were then fractionated by ammonium sulfate precipitation. The 60-95% (for HSP18.1) and 40-70% (for HSP17.7) ammonium sulfate fractions were dialyzed against 25 mM Tris-HCl, 1 mM EDTA, pH 7.5 (buffer B) and separated on 0.2-0.8 M linear sucrose gradients in buffer B for 2 h, 45 min at 50,000 rpm in a Beckman VTi50 rotor. Fractions containing the smHSPs were pooled and applied to a diethylaminoethyl-Sepharose Fast Flow column equilibrated with buffer B supplemented with 3 M urea. Small HSPs were eluted with a 0-0.2 M NaCl gradient in buffer B supplemented with 3 M urea and then dialyzed extensively against buffer B. SDS-PAGE indicated that the smHSP preparations were at least 95% pure (see Fig. 1). SDS-PAGE was performed using a 12.5% acrylamide gel as described (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Non-denaturing pore exclusion PAGE was performed on a 4-22.5% acrylamide gel according to the method of Helm et al.(15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar). Small HSPs were applied to a carbon film, rinsed with phosphate-buffered saline, and negatively stained with 1% uranyl acetate. The samples were viewed in a Phillips EM410 electron microscope operating at 80 kV. Micrographs were taken at × 55,000 magnification. Molecular weights of HSP18.1 and HSP17.7 complexes were determined at 4°C by sedimentation equilibrium in a Beckman Optima XLA analytical ultracentrifuge equipped with absorbance optics. A Beckman An60Ti rotor was used with six-sector equilibrium centerpieces at speeds ranging from 7,000 to 15,000 rpm. Centrifugation of HSP18.1 was carried out in buffer B, and centrifugation of HSP17.7 was carried out in buffer B supplemented with 50 mM NaCl. All samples were clarified at 40,000 × g before centrifugation. Solvent densities were calculated from buffer and protein composition, respectively, as described by Laue et al.(33Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (776) Google Scholar). Multiple data sets of A280 versus radial position were simultaneously fit to the Yphantis equation using a nonlinear least squares fit with the program HID4000 (34Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge1992: 90-125Google Scholar). The residuals were examined for aggregation and/or non-ideality. Analysis of the residuals for HSP18.1 indicated a homogenous solution, whereas analysis of the residuals for HSP17.7 indicated aggregation in some of the samples. These HSP17.7 data sets were deleted from the pool, and the remainder of the data sets was reanalyzed, resulting in better residuals. The molecular weights calculated with the edited and unedited data sets were close to each other (less than 5% difference), but the error was smaller with the edited sets. CS or LDH was denatured at a concentration of 1.5 μM in 6 M guanidine hydrochloride, 2 mM dithiothreitol, 100 mM Tris-HCl, pH 8.0 (for CS) or pH 7.5 (for LDH) for 1.5 h at 25°C. Refolding was initiated by diluting 2.5 μl of denatured CS or LDH into 247.5 μl of rapidly vortexing refolding buffer in 1.5-ml microcentrifuge tubes as described (35Wang Z. Landry S.J. Gierasch L.M. Srere P.A. Protein Sci. 1992; 1: 522-529Crossref PubMed Scopus (42) Google Scholar). The final refolding conditions were 150 nM CS or LDH in 100 mM Tris-HCl, pH 8.0 (for CS) or pH 7.5 (for LDH), 20 μM dithiothreitol, 2 mM (NH4)2SO4 carried over from the commercial enzyme stocks, and variable amounts of HSP18.1, HSP17.7, catalase, or lysozyme at 25°C. At the times indicated, 25-μl aliquots were assayed for CS or LDH activity. 150 nM CS or LDH was combined with varying amounts of HSP18.1 or HSP17.7 in 50 mM HEPES-KOH, pH 7.5 (total volume, 1 ml) in covered quartz cuvettes at 25°C. Where indicated, catalase or IgG was added at the concentrations indicated. Samples were incubated in a water bath at 45°C (for CS) or 55°C (for LDH) and then monitored for light scattering at 320 nm (36Jaenicke R. Rudolph R. Creighton T.E. Protein Structure: A Practical Approach. IRL Press, Oxford1989: 191-223Google Scholar) at the times indicated. 150 nM CS was incubated in the absence or presence of 150 nM HSP18.1, HSP17.7, 32 μg/ml catalase, or 50 μg/ml lysozyme in 50 mM HEPES-KOH, pH 8.0 in covered glass tubes (total volume, 0.5 ml) at the indicated temperatures. At various time points 25 μl aliquots were removed and measured for CS activity. All assays were performed at 25°C. CS activity was measured according to the method of Ochoa (37Ochoa S. Biochem. Prep. 1957; 5: 19-21Google Scholar) in which the disappearance of acetyl CoA is monitored by the decrease in absorbance at 233 nm. LDH activity was measured as described (38Kornberg A. Methods Enzymol. 1955; 1: 441-443Crossref Scopus (619) Google Scholar). The concentrations of smHSPs and LDH were determined using the Bio-Rad protein assay with bovine serum albumin as the standard. The concentration of CS was determined spectrophotometrically at 280 nm (39Bloxham D.P. Ericsson L.H. Titani K. Walsh K.A. Neurath H. Biochemistry. 1980; 19: 3979-3985Crossref PubMed Scopus (27) Google Scholar). The concentrations of smHSPs indicated in the text refer to the complexes composed of 12 subunits. Therefore, 150 nM smHSP is equivalent to approximately 32 μg/ml. The concentrations of CS and LDH refer to monomers. Errorbars in figures represent standard errors generated from at least three replicate trials. Recombinant smHSPs were purified from E. coli cells to greater than 95% homogeneity as soluble, high molecular weight complexes (Fig. 1). One liter of cell culture yielded approximately 6-10 mg of purified protein. Analysis of the protein preparations by non-denaturing pore exclusion PAGE indicated that the recombinant proteins, similar to those from heat-stressed pea roots (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar),2( 2K. Helm, G. Lee, and E. Vierling, manuscript in preparation. ) migrated exclusively as complexes with apparent molecular masses of 240 and 320 kDa for HSP18.1 and HSP17.7, respectively. Analysis of HSP18.1 by sedimentation equilibrium analytical ultracentrifugation yielded a molecular mass of 216,418 ± 6,601 Da, consistent with the apparent molecular size derived from non-denaturing PAGE. This molecular mass corresponds to 11.97 ± 0.37 HSP18.1 subunits, indicating that the native HSP18.1 oligomer consists of 12 subunits. Similar analysis revealed that HSP17.7 had a molecular size of 200,183 ± 8,208 Da, which corresponds to 11.28 ± 0.46 subunits, suggesting that HSP17.7, like HSP18.1, may also be composed of 12 subunits. Since migration of proteins on non-denaturing pore exclusion PAGE is dependent on both size and shape (40Anderson L.O. Borg H. Mikaelsson M. FEBS Lett. 1972; 20: 199-202Crossref PubMed Scopus (150) Google Scholar), it appears that HSP17.7 may differ structurally from HSP18.1 by virtue of shape. Indeed, electron microscopy of negatively stained HSP18.1 complexes at a magnification of 55,000 suggested that HSP18.1 forms uniformly globular particles with approximate diameters of 10 nm (Fig. 2 A). In contrast, similar analysis of HSP17.7 complexes revealed both round and triangular structures (Fig. 2 B), which may represent different orientations of the complexes. In Vitro Function of HSP18.1 and HSP17.7 Both HSP18.1 and HSP17.7 increased the folding yields of CS (homodimer of 50-kDa subunits) and LDH (tetramer of 35-kDa subunits), which had been denatured in 6 M guanidine hydrochloride. When 150 nM HSP18.1 or 240 nM HSP17.7 was added to 150 nM denatured CS monomers under conditions that stimulated refolding, greater than 40% (relative to an equivalent amount of non-denatured CS) attained the native state after 60 min (Fig. 3 A). In contrast, only 17% of the CS activity was spontaneously reactivated in the absence of the smHSPs. Although the smHSPs increased the yield of refolded CS, no rate enhancements were observed in their presence. Similar results were obtained when LDH was used as the target protein (Fig. 3 B). As a control, bovine catalase, a protein with a similar native molecular mass and isoelectric point (247 kDa, pI = 5.8) to HSP18.1 (217 kDa, pI = 5.96 (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar)) was substituted for the smHSPs. Addition of 32 μg/ml catalase, the equivalent weight-to-volume concentration of 150 nM smHSP, resulted in less than 18% reactivation of either CS or LDH, demonstrating that the enhancement in refolding was specific to the smHSPs (see Fig. 5). Adding 50 μg/ml lysozyme or further increasing the concentration of catalase to 128 μg/ml did not increase refolding yields (not shown).Figure 5:ATP or GTP does not affect smHSP-enhanced CS or LDH refolding. CS (A) or LDH (B) was denatured and allowed to refold as described in the legend to Fig. 4. Refolding reactions were carried out in the absence of any additions (Unassisted), in the presence of 32 μg/ml catalase, or in the presence of 150 nM HSP18.1 or HSP17.7, as indicated. Where indicated, reactions included 3 mM ATP or GTP supplemented with 6 mM MgCl2 and 50 mM KCl.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The refolding of CS and LDH was next examined as a function of the amount of smHSP in the refolding reactions. For both CS and LDH, refolding yields saturated at an smHSP complex-to-CS or -LDH monomer ratio of approximately 1 (Fig. 4). The observed enhancement of refolding by the smHSPs occurred in a nucleotide triphosphate-independent manner; addition of MgATP or MgGTP did not further stimulate refolding yields (Fig. 5). Since HSP18.1 and HSP17.7 are coordinately expressed in various tissues2(24DeRocher A.E. Vierling E. Plant J. 1994; 5: 93-102Crossref Scopus (106) Google Scholar), a mixture of the two proteins was evaluated for its ability to refold chemically denatured LDH. The amount of LDH reactivation observed for a mixture of 30 nM HSP18.1 plus 30 nM HSP17.7 was approximately equal to that observed in the presence of 60 nM (nonsaturating amounts according to Fig. 4) of either species alone (Fig. 6). These results suggest that HSP18.1 and HSP17.7 function independently of one another rather than synergistically in these assays. When heated to 45°C, CS began to form insoluble aggregates that could be detected by light scattering (Fig. 7). Aggregation, however, could be reduced in the presence of the smHSPs and was completely suppressed at an HSP18.1-to-CS ratio of 0.5 (Fig. 7 A). Catalase added to CS at a concentration of 32 μg/ml had no protective effect; nor did 50 μg/ml lysozyme (not shown). HSP17.7 also prevented CS thermal aggregation but was less effective than HSP18.1; only 65% of CS scattering could be prevented upon addition of HSP17.7 at an HSP17.7-to-CS ratio of 1.6 (Fig. 7 B). Beyond this, increasing amounts of HSP17.7 had no further protective effect. Under these conditions, neither HSP18.1 nor HSP17.7 alone gave rise to any detectable light scattering (not shown). Light scattering by LDH could not be detected at 45°C but was observed when the temperature was increased to 55°C. At this higher temperature HSP18.1 was less effective against LDH aggregation as compared with CS aggregation at 45°C (Fig. 8). At a concentration of 150 nM LDH monomers, the protective effect of HSP18.1 saturated at a concentration of 600 nM HSP18.1 complex. However, addition of 128 μg/ml bovine IgG or lysozyme (not shown), both of which are stable at 55°C, had no protective effect, suggesting the level of protection by HSP18.1 was significant. HSP17.7 aggregated itself at 55°C, thereby preventing further analysis (not shown). When CS was incubated at 38°C alone or in the presence of 32 μg/ml catalase, less than 5% of the original CS activity remained after 60 min (Fig. 9). Upon temperature shift of the samples to 22°C, less than 15% of the original CS activity remained after 60 min in the presence or absence of 32 μg/ml catalase. Similar results were obtained when 50 μg/ml lysozyme was substituted for catalase (not shown). In contrast, when CS was treated similarly but in the presence of stoichiometric amounts of HSP18.1 or HSP17.7, CS activity was minimally protected at 38°C, but approximately 65-70% of the original activity was subsequently regained after 60 min at 22°C. However, the thermal protective effect of the smHSPs was only observed if the smHSPs were present during the initial high temperature incubation; addition of smHSPs at the time of temperature shift to 22°C did not result in CS reactivation (Fig. 9). Although smHSPs protected CS against irreversible inactivation at 38°C, essentially no protection of activity was observed at 45°C in the presence of either HSP18.1 or HSP17.7 (Fig. 10) despite the fact that HSP18.1 efficiently suppressed CS aggregation at this temperature (Fig. 7). Furthermore, in the presence or absence of the smHSPs, no CS reactivation could be detected after shifting the samples from 45 to 22°C (not shown). From this and other recent studies of a variety of plant smHSPs, it appears that smHSPs from plant sources generally form oligomeric complexes ranging in size from 200 to 240 kDa (4Arrigo A.-P. Landry J. Morimoto R. Tissieres A. Georgeopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, NY1994: 335-373Google Scholar, 15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar, 16Helm K.W. LaFayette P.R. Nagao R.T. Key J.L. Vierling E. Mol. Cell. Biol. 1993; 13: 238-247Crossref PubMed Scopus (77) Google Scholar, 17Chen Q. Osteryoung K. Vierling E. J. Biol. Chem. 1993; 269: 13216-13223Abstract Full Text PDF Google Scholar). Here, we have characterized two representatives of plant cytosolic smHSPs from P. sativum, HSP18.1 and HSP17.7. Both behave solely as oligomers composed of 12 subunits. Since most other plant smHSPs have native molecular sizes similar to those of HSP18.1 and HSP17.7, as judged by non-denaturing PAGE, it is likely that other plant smHSPs are also composed of 12 subunits. A structural model consisting of an inner core of 12 subunits has been previously proposed for α-crystallin high molecular weight complexes (41Tardieu A. Laporte D. Licinio P. Krop B. Delaye M. J. Mol. Biol. 1986; 192: 711-724Crossref PubMed Scopus (140) Google Scholar). In contrast, smHSP complexes from mammalian sources have been found to exist in larger, more dynamic complexes. For example, the murine smHSP, HSP25, has been characterized as a 32-mer with a native molecular mass of 730 kDa (19Behlke J. Lutsch G. Gaestel M. Bielka H. FEBS Lett. 1991; 288: 119-122Crossref PubMed Scopus (59) Google Scholar). Recent evidence suggests that the subunit composition of mammalian smHSPs can be highly variable in response to phosphorylation state (20Kato K. Hasegawa K. Goto S. Inaguma Y. J. Biol. Chem. 1994; 269: 11274-11278Abstract Full Text PDF PubMed Google Scholar, 21Benndorf R. Hayeβ K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar) and that the control of smHSP oligomerization may regulate their interaction with actin (21Benndorf R. Hayeβ K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). At present however, phosphorylation of plant small HSPs has not been detected (22Nover L. Scharf K.-D. Eur. J. Biochem. 1984; 139: 303-313Crossref PubMed Scopus (100) Google Scholar), and the recognition sites for phosphorylation observed in mammalian smHSPs (41Tardieu A. Laporte D. Licinio P. Krop B. Delaye M. J. Mol. Biol. 1986; 192: 711-724Crossref PubMed Scopus (140) Google Scholar, 42Gaestel M. Schroder W. Benndorf R. Lippmann C. Buchner K. Hucho F. Erdmann V.A. Bielka H. J. Biol. Chem. 1991; 266: 14721-14724Abstract Full Text PDF PubMed Google Scholar) have not been conserved in plant smHSPs (23Plesovsky-Vig N. Vig J. Brambl R. J. Mol. Evol. 1992; 35: 537-545Crossref PubMed Scopus (59) Google Scholar). Although HSP18.1 and HSP17.7 share common features such as 60% amino acid similarity (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar), similar subunit size, and native molecular weight as determined by analytical ultracentrifugation, their different mobilities on non-denaturing pore exclusion PAGE suggest that other structural differences may distinguish class I and class II smHSPs. While the relative migration of HSP18.1 (compared to globular standards) was consistent with that predicted by analytical ultracentrifugation, the migration of HSP17.7 was inconsistent. Based on these findings, it is likely that the shape of the HSP17.7 complex deviates from a globular structure. This observation would be consistent with the triangular structures seen exclusively in HSP17.7 preparations by electron microscopy. Such a large structural difference between HSP18.1 and HSP17.7 may account for the fact that mixed complexes between the two species are not observed in vivo and will not form in vitro.2 Indicative of a molecular chaperone function for HSP18.1 and HSP17.7, both proteins were able to influence the folding and aggregation properties of model substrates during several in vitro assays. As has been previously reported for mammalian smHSPs and α-crystallins (9Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1726) Google Scholar, 11Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar), refolding of chemically denatured proteins could be enhanced with the addition of stoichiometric levels of HSP18.1 or HSP17.7 in an ATP-independent manner. Whereas murine HSP25 maximally enhanced the refolding of chemically denatured CS at an HSP25 complex-to-CS monomer ratio of 1:3, the enhancement by the plant smHSPs tested here saturated at a ratio of approximately 1:1. Since the native molecular mass of HSP25 has been estimated at 730 kDa and those for HSP18.1 and HSP17.7 have been estimated between 200 and 216 kDa, the plant smHSPs appear to be as efficient as the mammalian smHSPs on a per weight basis. An additional consideration is that in the present study, we have utilized 6 M guanidine HCl as the protein denaturant, as opposed to 8 M urea, based on the finding that 8 M urea is insufficient to produce CS monomers (43Landry J. Lambert H. Zhou M. Lavoie J.N. Hickey E. Weber L.A. Anderson C.W. J. Biol. Chem. 1992; 267: 794-803Abstract Full Text PDF PubMed Google Scholar). Therefore, a precise stoichiometric comparison between the previous and the present study may be difficult based on the different oligomerization and folding states adopted by CS after guanidine versus urea denaturation. Nonetheless, it appears that the functional properties among plant and mammalian smHSPs have been conserved despite the fact the two groups share only limited overall sequence homology. The ability of HSP18.1 and HSP17.7 to prevent thermal aggregation of model substrates provides further support for their putative role in thermotolerance. Complete suppression of CS aggregation by HSP18.1 at 45°C was observed at an HSP18.1 complex-to-CS monomer ratio of 0.5:1, although HSP17.7 was far less efficient. By comparison, similar, maximal protection by murine HSP25 was observed at a ratio of 0.67:1 (11Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar). Since under these conditions CS dimers would be expected to prevail, the true complex-to-substrate stoichiometry would be approximately 1:1 for both HSP18.1 and HSP25. Less efficient protection by HSP18.1 or lack of protection by HSP17.7 imparted to LDH at 55°C suggests that this temperature may exceed the functional temperature range for these smHSPs. However, the temperatures necessary to induce insolubilization of CS (45°C) and LDH (55°C) exceed the temperature of maximum smHSP synthesis in plants (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar) as well as the heat stress temperatures generally experienced by plants under field conditions (44Hernandez L. Vierling E. Plant Physiol. 1993; 101: 1209-1216Crossref PubMed Scopus (68) Google Scholar). We sought to evaluate the protective effects of HSP18.1 and HSP17.7 at 38°C, the temperature at which these smHSPs are maximally induced (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar). By employing a more physiologically relevant elevated temperature of 38°C followed by a return to 22°C, we found that both HSP18.1 and HSP17.7 displayed similar behavior in protecting CS from irreversible heat inactivation. The predominant effect of the smHSPs was to increase the potential of heat-inactivated CS to properly refold at the permissive temperature of 22°C. These findings suggest that in vivo the presence of HSP18.1 and HSP17.7 may have important consequences during both heat stress and recovery and are consistent with the observation that HSP18.1 and other smHSPs remain abundant in tissues during and after heat stress (15DeRocher A.E. Helm K.W. Lauzon L.M. Vierling E. Plant Physiol. 1991; 96: 1038-1047Crossref PubMed Scopus (117) Google Scholar). The fact that the protective effects of the smHSPs could only be observed if the smHSPs were present during the 38°C incubation indicates that the smHSPs must be present during the time of thermal denaturation when irreversible misfolding and/or aggregation are most likely to occur. These findings are similar to those described for heat-inactivated firefly luciferase, which can be subsequently reactivated by incubation at room temperature in the presence of the chaperones DnaK, DnaJ, and GrpE (45Schroeder H. Langer T. Hartl F.-U. Bukau B. EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (496) Google Scholar). At minimum, the presence of DnaJ is required during thermal inactivation to prevent aggregation of luciferase, whereas reactivation requires the addition of DnaK and GrpE. Unlike the HSP60, HSP70, and TriC classes of molecular chaperones (12Hendrick J.P. Hartl F.-U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1457) Google Scholar), the in vitro smHSP functional cycle does not require ATP or other factors that regulate the release of substrates bound to the chaperone. Thus, it is probable that smHSPs recognize non-native substrates but bind them reversibly. By this mechanism, smHSPs may lower the effective concentration of free, unfolded intermediates and reduce off-pathway processes such as aggregation or misfolding. Pools of free substrates could then spontaneously refold under permissive conditions. This relatively passive mechanism, which requires neither an energy source nor coordination with other proteins, may explain why refolding of chemically denatured CS is relatively less efficient in the presence of the smHSPs than in the presence of GroEL, GroES, and ATP. For example, up to 80% CS reactivation has been observed in the presence of saturating amounts (6-fold molar excess) of the GroE system (35Wang Z. Landry S.J. Gierasch L.M. Srere P.A. Protein Sci. 1992; 1: 522-529Crossref PubMed Scopus (42) Google Scholar), compared with 45% in the presence of saturating (stoichiometric) amounts of smHSPs. However, it is highly unlikely that the enhancements in refolding of chemically denatured CS or LDH observed in the present study resulted from nonspecific solute effects since excesses of the control proteins catalase or lysozyme did not stimulate refolding. These results imply that the smHSPs were not simply reducing the number of molecular collisions between aggregation-prone folding intermediates. The failure of the smHSPs to facilitate reactivation of CS incubated at 45°C may be indicative of irreversible CS misfolding that cannot be prevented even by association with the smHSPs at this higher temperature. Under such conditions, however, smHSP-promoted solubilization of permanently damaged proteins, like that observed with CS in the presence of HSP18.1, may facilitate degradation by proteolytic systems. Such a role has been proposed for other molecular chaperones (46Parsell D.A. Lindquist S. Annu. Rev. Genet. 1994; 27: 437-496Crossref Scopus (1850) Google Scholar, 47Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar). The in vivo function of HSP18.1 and HSP17.7 is not understood at present; nor have any endogenous substrates of these smHSPs been identified. The present study suggests that both HSP18.1 and HSP17.7 can recognize and interact with different foreign proteins that possess non-native structures as the common feature. In this light, HSP18.1 and HSP17.7 may function as nonspecific chaperones. However, in vivo, more specific protein-protein interactions may occur and may be highly specific for either class I or class II proteins. Such specialization may account for the strong evolutionary conservation of these two classes of cytosolic smHSPs in higher plants. We thank Jörg Thierfelder for providing the Pshsp18.1 and Pshsp17.7 cDNAs subcloned into pJC20 and Dr. Paul Matsudaira for performing the electron microscopic analysis of the smHSPs. We also thank James Grille for expert photographic assistance and Dr. Katherine Osteryoung for critical reading of this manuscript." @default.
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• W2022038675 title "Structure and in Vitro Molecular Chaperone Activity of Cytosolic Small Heat Shock Proteins from Pea" @default.
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