Protein J (2013) 32:449–455 DOI 10.1007/s10930-013-9504-3
The RNA Chaperone and Protein Chaperone Activity of Arabidopsis Glycine-Rich RNA-Binding Protein 4 and 7 is Determined by the Propensity for the Formation of High Molecular Weight Complexes Ji Hoon Han • Young Jun Jung • Hyun-Ju Lee • Hyun Suk Jung • Kyun Oh Lee • Hunseung Kang
Published online: 3 August 2013 Ó Springer Science+Business Media New York 2013
Abstract RNA chaperones and protein chaperones are cellular proteins that can aid the correct folding of target RNAs and proteins, respectively. Although many proteins possessing RNA chaperone or protein chaperone activity have been demonstrated in diverse organisms, report evaluating the RNA chaperone and protein chaperone activity of a given protein is severely limited. Here, two glycine-rich RNA-binding proteins in Arabidopsis thaliana (AtGRPs), AtGRP7 exhibiting RNA chaperone activity and AtGRP4 exhibiting no RNA chaperone activity, were investigated for their protein chaperone activity. The heatinduced thermal aggregation of a substrate protein was significantly decreased with the addition of AtGRP4 depending on protein concentration, whereas the thermal aggregation of a substrate protein was further increased with the addition of AtGRP7, demonstrating that AtGRP4 but not AtGRP7 possesses protein chaperone activity. Size exclusion chromatography and electron microscopy analyses revealed that the formation of high molecular weight (HMW) complexes is closely related to the protein chaperone activity of AtGRP4. Importantly, the additional 25 amino acids at the N-terminus of AtGRP4 are crucial for J. H. Han H. Kang (&) Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Kwangju 500-757, Korea e-mail:
[email protected] Y. J. Jung K. O. Lee Division of Applied Life Science (BK21 Program) and PMBBRC, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Korea H.-J. Lee H. S. Jung Division of Electron Microscopic Research, Korea Basic Science Institute, 113 Gwahangno, Yuseong-gu, Taejon 305-333, Korea
HMW complex formation and protein chaperone activity. Taken together, these results show that the formation of HMW complexes is important for determining the RNA chaperone and protein chaperone activity of AtGRP4 and AtGRP7. Keywords Arabidopsis thaliana Glycine-rich RNA-binding protein Protein chaperone Protein folding RNA chaperone RNA folding Abbreviations AtGRP4 Arabidopsis thaliana GRP4 AtGRP7 Arabidopsis thaliana GRP7 GRP Glycine-rich RNA-binding protein HMW High molecular weight MDH Malate dehydrogenase
1 Introduction Correct folding of RNAs and proteins into native conformations is essential for cellular functions of these biomolecules in living organisms. During RNA folding process, RNA molecules have intrinsic thermodynamic and kinetic folding problems, in which RNA molecules are trapped in misfolded structures and frequently misfold into structurally stable but biologically inactive structures [8]. During protein folding process, proteins are also misfolded into biologically inactive structures, which results in the aggregation of unfolded proteins. To counter these adverse effects resulted from RNA and protein misfolding and to overcome folding problems, many chaperone proteins have evolved, which promote the formation of native RNA or protein folds.
123
450
Molecular chaperones are proteins that assist proper protein folding by preventing aggregation or by directing correct folding process in cells. In order to differentiate it from the proteins with RNA chaperone activity, we will designate molecular chaperone as protein chaperone in this paper. Protein chaperones are mainly classified into 2 groups: holdase chaperones that prevent nonspecific aggregation by binding to folding intermediates and foldase chaperones that assist the folding of denatured intermediates to their native states [3]. The proteins with RNA chaperone activity are nonspecific RNA-binding proteins that can disrupt base pairs in diverse RNA substrates and aid the folding of RNA substrates [8, 24]. Many proteins present in prokaryotes and eukaryotes have been determined to have RNA chaperone function during diverse cellular processes [12, 13]. Although most chaperone proteins play roles in the folding of either RNAs or proteins, some proteins are involved in the folding of both RNAs and proteins. The term ‘‘Janus chaperone’’ was first proposed to represent chaperone proteins that aid the folding of both RNAs and proteins [18], which reflects the term ‘‘Janus kinases’’ in the field of protein phosphorylation [23]. However, reports demonstrating the chaperone proteins possessing both RNA chaperone and protein chaperone activity are severely limited. It has been demonstrated that many large ribosomal subunit proteins in Escherichia coli possess RNA chaperone activity [25]. In addition, four large ribosomal subunit proteins, L15, L16, L18, and L19, have been determined to contain protein chaperone activity as well [18]. The nuclear protein nucleolin has also been determined to possess both protein and RNA chaperone activities [1, 2]. Although many proteins possessing either RNA chaperone or protein chaperone activity have been determined in different organisms, report evaluating the RNA chaperone and protein chaperone activity of a given protein is severely limited. Plants contain diverse RNA-binding proteins, among which those harboring RNA-recognition motif at the N-terminal half and a glycine-rich region at the C-terminal half are referred to as glycine-rich RNA-binding proteins (GRPs). Plant GRPs have been demonstrated to play important roles in stress response by functioning as RNA chaperones [13]. Here, we aimed at comparing the protein chaperone and RNA chaperone activity of the two Arabidopsis thaliana GRPs, AtGRP7 and AtGRP4, because the two GRPs are strikingly different in their RNA chaperone activities and functions. It has been demonstrated that AtGRP7 but not AtGRP4 possesses RNA chaperone activity and confers freezing tolerance in Arabidopsis [15, 16, 19]. By directly comparing the RNA chaperone and protein chaperone activities of AtGRP4 and AtGRP7, we show that the formation of high molecular weight (HMW) complexes is important for determining the RNA chaperone and protein chaperone activity of AtGRP4 and AtGRP7.
123
J. H. Han et al.
2 Materials and Methods 2.1 Recombinant Protein Purification The cDNAs encoding native A. thaliana AtGRP4 (At3g23830) and AtGRP7 (At2g21660) proteins and the N-terminal-deleted AtGRP4 protein (AtGRP4-25N) were cloned into the pGEX4T-3 vector (Amersham Pharmacia Biosciences). The AtGRP4-25N was constructed by deleting the N-terminal 25 amino acids from AtGRP4 and the chimeric AtGRP4N7C was constructed by ligating the N-terminal region of AtGRP4 (from amino acid 1 to 122) to the C-terminal region of AtGRP7 (from amino acid 88 to 177). The detailed procedures to generate the constructs have been described in the previous report [20]. For the expression and purification of recombinant glutathione S-transferase-AtGRP proteins in E. coli, the constructs were transformed into BL21 DE3 competent cells (Promega), and the recombinant proteins were purified with glutathione Sepharose 4B resin. The glutathione S-transferase was removed from the recombinant fusion proteins by thrombin digestion, and the purified proteins were used for subsequent analysis. 2.2 Protein Chaperone Activity Assay Holdase chaperone activity of the proteins was assessed by measuring its capacity to suppress thermal aggregation of malate dehydrogenase (MDH) as an in vitro substrate essentially as previously described [21]. Heat-induced thermal aggregation of MDH was initiated by the addition of the purified proteins in 50 mM Hepes-KOH (pH 8.0) at 45 °C. The turbidity due to substrate aggregation was monitored by measuring the absorbance at 340 nm in a temperature-controlled spectrophotometer (DU800, Beckman). 2.3 Size Exclusion Chromatography Size exclusion chromatography on FPLC (AKTA, Amersham Biosciences, Sweden) was performed with Superdex 200 HR column equilibrated with 50 mM Hepes (pH 8.0) buffer containing 150 mM NaCl. Proteins were eluted with the same buffer at a flow rate of 0.5 ml/min as previously described [10, 21]. The amounts of the eluted protein complexes at each time point were monitored by measuring the absorbance at 280 nm. 2.4 Electron Microscopy The purified AtGRP proteins were dissolved in 50 mM Hepes (pH 8.0) containing 150 mM NaCl. Five microliter of the protein solution was applied to the carbon-coated grid that was glow-discharged for 3 min in air, and the
RNA Chaperone and Protein Chaperone Activity of GRPs
451
specimen was negatively stained using 1 % uranyl acetate as previously described [11]. The grids were examined in a Technai G2 Spirit Twin transmission electron microscope (FEI, USA) operated at 120 kV, and the images were recorded on an Ultrascan 895 CCD camera (Gatan, USA).
3 Results 3.1 AtGRP4 but not AtGRP7 Exhibits Protein Chaperone Activity AtGRP4 and AtGRP7 harbor 136 amino acids and 177 amino acids, respectively (Fig. 1a). Amino acid sequence comparison showed that AtGRP4 contains additional N-terminal amino acids, while AtGRP7 harbors additional glycine-rich region at the C-terminus (Fig. 1a). Structure prediction by molecular modeling revealed that the conserved regions of AtGRP4 and AtGRP7 adopt highly similar structures consisting of two a-helices and four
b-strands. However, the additional N-terminal amino acids present in AtGRP4 and the C-terminal glycine-rich regions found in AtGRP4 and AtGRP7 adopt disordered structures (Fig. 1b). It has been determined in our previous analysis that AtGRP7 possesses RNA chaperone activity, whereas AtGRP4 does not possess RNA chaperone activity [16, 20]. To evaluate whether AtGRP7 and AtGRP4 possess protein chaperone function, the recombinant AtGRP4 and AtGRP7 proteins were expressed and purified in E. coli and their holdase chaperone activity was analyzed using MDH as a substrate essentially as previously described [21]. When AtGRP4 was added into the reaction mixture containing MDH, the light scattering of the solution decreased depending on protein concentration, whereas the light scattering of the reaction solution adding AtGRP7 increased depending on protein concentration (Fig. 2). These results indicate that AtGRP4 harboring no RNA chaperone activity exhibits protein chaperone function, whereas AtGRP7 possessing RNA chaperone activity does not exhibit protein chaperone function.
a
RNP2
RNP1
b
N
AtGRP4 (violet) AtGRP7 (white)
N
Fig. 1 Sequence comparison and predicted structures of AtGRP4 and AtGRP7. a Amino acid sequence alignment was made using the ClustalW program. Gaps in the sequences are indicated by dashes. The positions of ribonucleoprotein1 (RNP1) and RNP2 are indicated by red boxes, and the positions of predicted a-helices (H) and b-strands (E) are indicated above the sequences. The 25 amino acids
C
C
at the N-terminus of AtGRP4 are boxed. b Structural modeling of AtGRP4 and AtGRP7. The AtGRP4 and AtGRP7 structures were predicted using the program PS2 (http://ps2.life.nctu.edu.tw) with the structure of A18 hnRNP (PDB: 1X5S) as a template. The structures are viewed using UCSF Chimera software (Color figure online)
123
452
J. H. Han et al.
solution increased depending on protein concentration (Fig. 2). These results demonstrate that AtGRP4-25N does not possess protein chaperone activity, indicating that the 25 amino acids present at the N-terminus of AtGRP4 are indispensible for protein chaperone activity. 3.3 Formation of High Molecular Weight Complexes is Important for Protein Chaperone activity
Fig. 2 The holdase chaperone activity of AtGRPs. The purified AtGRPs were mixed with malate dehydrogenase (MDH) as a substrate, and the degree of heat-induced aggregation was quantified by measuring the absorbance at 340 nm in a spectrophotometer at 45 °C. The ratios between MDH and AtGRP4, AtGRP7, or AtGRP425N in the reaction mixture were indicated in the parenthesis
3.2 The N-terminal extension of AtGRP4 is indispensible for protein chaperone activity Amino acid sequence comparison showed that AtGRP4 contains additional N-terminal 25 amino acids which are not present in AtGRP7 (Fig. 1), and it has been demonstrated that this N-terminal extension is responsible for the lack of RNA chaperone activity of AtGRP4 [20]. To determine whether this N-terminal extension found in AtGRP4 affects protein chaperone activity of the protein, the recombinant AtGRP4 lacking the N-terminal extension (AtGRP4-25N) was purified and its holdase chaperone activity was analyzed. When AtGRP4 was incubated with MDH in the reaction solution, the light scattering of the solution decreased in a concentration dependent manner, whereas when AtGRP4-25N was incubated with MDH in the reaction solution, the light scattering of the protein
123
It has been demonstrated that the foldase and holdase chaperone functions depend on the formation of high molecular weight (HMW) complexes [21]. To determine whether the protein chaperone activity of AtGRP4 is related to its propensity for the formation of HMW complexes, the molecular sizes of protein complexes were analyzed by size exclusion chromatography and electron microscopy. It was evident from size exclusion chromatography analysis that AtGRP4 possessing protein chaperone activity forms HMW complexes, whereas AtGRP7 that has no protein chaperone activity does not form HMW complexes (Fig. 3a). Importantly, the AtGRP4-25N that lacked the N-terminal extension of AtGRP4 and exhibited no protein chaperone function did not form HMW complexes (Fig. 3a). To further confirm whether AtGRP4 or AtGRP7 forms HMW complexes, the molecular sizes of protein complexes were examined by electron microscopy analysis. It was evident that AtGRP4 possessing protein chaperone activity forms HMW complexes, whereas AtGRP7 that harbors no protein chaperone activity does not form HMW complexes (Fig. 3b). Importantly, the AtGRP4-25N that exhibited no protein chaperone function did not form HMW complexes (Fig. 3b). All of these results clearly demonstrate that the protein chaperone activity of AtGRP4 depends on the formation of HMW complexes. Because the formation of HMW complexes is closely correlated with the protein chaperone activity of AtGRPs, we further probed the importance of HMW complex formation for protein chaperone activity by analyzing the chimeric AtGRP4N7C that was constructed by ligating the N-terminal region of AtGRP4 (from amino acid 1 to 122) to the C-terminal region of AtGRP7 (from amino acid 88 to 177). It was evident that addition of the chimeric AtGRP4N7C into the reaction mixture containing MDH increased the light scattering of the solution in a concentration-dependent manner (Fig. 4a), indicating that AtGRP4N7C does not exhibit protein chaperone function. Size exclusion chromatography and electron microscopy analyses showed that AtGRP4N7C does not form HMW complexes (Fig. 4b, c). These results further support the notion that the formation of HMW complexes is important for the protein chaperone activity of AtGRPs.
RNA Chaperone and Protein Chaperone Activity of GRPs
453
a
b
AtGRP7
AtGRP4
AtGRP4-25N
Fig. 3 Analysis of high molecular weight complexes of AtGRPs. a The protein complexes were separated via size exclusion chromatography using Superdex 200 HR column, and the amounts of the eluted protein complexes of AtGRP4, AtGRP7, and AtGRP4-25N at each time point were monitored by measuring the absorbance at 280 nm. b Electron microscopic analysis of high molecular weight
complexes of AtGRPs. The AtGRP4, AtGRP7, and AtGRP4-25N were applied on carbon-coated grids, and the complexes on the negatively stained fields were observed under TEM. Black and white arrows indicate individual complexes observed in each field. Bar 200 nm
4 Discussion
foldase chaperone and disulfide reductase activities in low molecular weight forms [21]. It has been demonstrated that Arabidopsis h-type Trx possesses dual activities, which exhibits disulfide reductase activity in low molecular weight forms and molecular chaperone activity in HMW complexes [22]. It has also been demonstrated that the cytosolic yeast peroxiredoxins, cPrx I and II, have peroxidase function in low molecular weight forms and chaperone function in HMW complexes [10]. Our current analyses together with these previous reports clearly demonstrate that the ability of the proteins to function as protein chaperones depends on its capability to form HMW complexes. It has been shown that the functional flexibility of a protein is correlated with the structural disorderness of the protein [26, 27]. The disordered regions of proteins are able to interact with diverse substrates, and the structural disorderness and adoptability are relevant with RNA chaperone and protein chaperone activities [6, 9, 17, 27]. The nuclear protein nucleolin adopts highly disordered structures, which is important for its Janus chaperone activity [1, 2]. Our analysis also suggests that the chaperone
The present study demonstrates that the RNA chaperone and protein chaperone activities of AtGRP4 and AtGRP7 are inversely related to each other. AtGRP7 that possesses RNA chaperone activity does not exhibit protein chaperone function, whereas AtGRP4 that harbors no RNA chaperone activity exhibits protein chaperone function. The ability of AtGRP4 to function as protein chaperone is related to its propensity for HMW complex formation. The relationship between protein chaperone activity and HMW complex formation has been previously demonstrated. Small heat shock proteins have been shown to exhibit molecular chaperone activity, which protects unfolded protein substrates from irreversible aggregation in vitro. High molecular weight complex of small heat shock proteins accompanied by much higher affinity for unfolded clients at heat shock temperatures is a prerequisite for efficient chaperone activity [5, 7]. The activity of Arabidopsis thioredoxin-like protein depends on its oligomeric status such that thioredoxin-like protein exhibits the holdase chaperone activity in HMW complexes, whereas it exhibits the
123
454
J. H. Han et al.
region of AtGRP4 adopts high disordered structures (Fig. 1) and that the additional 25 amino acids at the N-terminus of AtGRP4 interfere with RNA chaperone activity [20]. Clearly, the disordered peptide at the N-terminus of AtGRP4 was indispensible for HMW complex formation (Fig. 3), which is needed for the protein chaperone activity of AtGRP4. Notably, the chimeric AtGRP4N7C harboring the N-terminal AtGRP4 and the C-terminal AtGRP7 did not form HMW complexes and did not exhibit protein chaperone activity (Fig. 4). These results suggest that the sequences and arrangement of specific domains of AtGRP4 and AtGRP7 as well as the disordered peptide at the N-terminus of AtGRP4 determine HMW complex formation and protein chaperone function. The physiological significance of AtGRP4’s protein chaperone activity is not understood yet. The RNA-recognition motif is involved in the interaction of GRPs with RNA substrates, and it has been determined that AtGRP4 binds sequence-nonspecifically to RNAs and binds to singlestranded DNA and double-stranded DNA as well [19]. As the C-terminal glycine-rich regions of GRPs have been demonstrated to be involved in protein–protein interactions [4, 14], AtGRP4 is likely to bind proteins as well as RNAs. We propose that the formation of HMW complexes through the N-terminal extension and the interactions with diverse protein substrates through the C-terminal glycinerich region are important for AtGRP4’s protein chaperone activity. In conclusion, we show that the RNA chaperone and protein chaperone activities of AtGRP4 and AtGRP7 are determined by the propensity for the formation of HMW complexes of the proteins. Considering the limited information about specific amino acid sequences and structures necessary for RNA chaperone or protein chaperone activity of proteins, the current work provides new insights into the significance of domain sequences and structures of GRPs necessary for RNA chaperone and protein chaperone functions. More studies should be done to fully understand which structural features of a protein are important for RNA chaperone or protein chaperone functions.
Fig. 4 Analysis of the holdase chaperone activity and high molecular weight complexes of the chimeric AtGRP4N7C. a The purified AtGRP4N7C was mixed with malate dehydrogenase (MDH) as a substrate, and the degree of heat-induced aggregation was quantified by measuring the absorbance at 340 nm in a spectrophotometer at 45 °C. The protein complexes were analyzed via b size exclusion chromatography and c electron microscopy as described in Fig. 3. Black arrows indicate individual complexes. Bar 100 nm
Acknowledgments This work was supported by the grant from the Mid-career Researcher Program through the National Research Foundation of Korea grant funded by the Ministry of Education, Science and Technology (2011-0017357). EM works were performed in the EM facility of Korea Basic Science Institute (Grant No. KBSI T33415).
References activity of a protein is related with its structural disorderness. Our current and previous analyses showed that the N-terminal extension as well as the C-terminal glycine-rich
123
1. Angelov D, Bondarenko VA, Almagro S, Menoni H, Mongelard F, Hans F, Mietton F, Studitsky VM, Hamiche A, Dimitrov S, Bouvet P (2006) EMBO J 25:1669–1679
RNA Chaperone and Protein Chaperone Activity of GRPs 2. Allain FH, Bouvet P, Dieckmann T, Feigon J (2000) EMBO J 19:6870–6881 3. Beissinger M, Buchner J (1998) Biol Chem 379:245–259 4. Burd CG, Dreyfuss G (1994) Science 265:615–621 5. Eyles SJ, Gierasch LM (2010) Proc Natl Acad Sci USA 107:2727–2728 6. Fuxreiter M, Simon I, Friedrich P, Tompa P (2004) J Mol Biol 338:1015–1026 7. Haslbeck M, Walke S, Stromer T, Ehrnsperger M, White HE, Chen S, Saibil HR, Buchner J (1999) EMBO J 18:6744–6751 8. Herschlag D (1995) J Biol Chem 270:20871–20874 9. Ivanyi-Nagy R, Lavergne JP, Gabus C, Ficheux D, Darlix JL (2008) Nucleic Acids Res 36:712–725 10. Jang HH, Lee KO, Chi YH, Jung BG, Park SK, Park JH, Lee JR, Lee SS, Moon JC, Yun JW et al (2004) Cell 117:625–635 11. Jung HS, Komatsu S, Ikebe M, Craig R (2008) Mol Biol Cell 19:3234–3242 12. Jung HJ, Park SJ, Kang H (2013) J Plant Biol 56:123–129 13. Kang H, Park SJ, Kwak KJ (2013) Trends Plant Sci 18:100–106 14. Kenan DJ, Query CC, Keene JD (1991) Trends Biochem Sci 16:214–220 15. Kim JS, Jung HJ, Lee HJ, Kim KA, Goh CH, Woo Y, Oh SH, Han YS, Kang H (2008) Plant J 55:455–466 16. Kim JS, Park SJ, Kwak KJ, Kim YO, Kim JY, Song J, Jang B, Jung CH, Kang H (2007) Nucleic Acids Res 35:506–516
455 17. Kovacs D, Kalmar E, Torok Z, Tompa P (2008) Plant Physiol 147:381–390 18. Kovacs D, Rakacs M, Agoston B, Lenkey K, Semrad K, Schroeder R, Tompa P (2009) FEBS Lett 583:88–92 19. Kwak KJ, Kim YO, Kang H (2005) J Exp Bot 56:3007–3016 20. Kwak KJ, Park SJ, Han JH, Kim MK, Oh SH, Han YS, Kang H (2011) J Exp Bot 62:4003–4011 21. Lee JR, Lee SS, Jang HH, Lee YM, Park JH, Park SC, Moon JC, Park SK, Kim SY, Lee SY, Chae HB, Jung YJ, Kim WY, Shin MR, Cheong GW, Kim MG, Kang KR, Lee KO, Yun DJ (2009) Proc Natl Acad Sci USA 106:5978–5983 22. Park SK, Jung YJ, Lee JR, Lee YM, Jang HH, Lee SS, Park JH, Kim SY, Moon JC, Lee SY, Chae HB, Shin MR, Jung JH, Kim MG, Kim WY, Yun D-J, Lee KO, Lee SY (2009) Plant Physiol 150:552–561 23. Podder H, Kahan BD (2004) Expert Opin Ther Targets 8:613–629 24. Rajkowitsch L, Chen D, Stampfl S, Semrad K, Waldsich C, Mayer O, Jantsch MF, Konrat R, Bla¨si U, Schroeder R (2007) RNA Biol 4:118–130 25. Semrad K, Green R, Schroeder R (2004) RNA 10:1855–1860 26. Tompa P, Csermely P (2004) FASEB J 18:1169–1175 27. Tompa P, Szasz C, Buday L (2005) Trends Biochem Sci 30:484–489
123
