Characterization of AtBAG2 as a Novel Molecular Chaperone

Bcl-2-associated anthanogene (BAG) family proteins regulate plant defense against biotic and abiotic stresses; however, the function and precise mechanism of action of each individual BAG protein are not yet clear. In this study, we investigated the biochemical and molecular functions of the Arabidopsis thaliana BAG2 (AtBAG2) protein, and elucidated its physiological role under stress conditions using mutant plants and transgenic yeast strains. The T-DNA insertion atbag2 mutant plants were highly susceptible to heat shock, whereas transgenic yeast strains ectopically expressing AtBAG2 exhibited outstanding thermotolerance. Moreover, a biochemical analysis of GST-fused recombinant proteins produced in bacteria revealed that AtBAG2 exhibits molecular chaperone activity, which could be attributed to its BAG domain. The relevance of the molecular chaperone function of AtBAG2 to the cellular heat stress response was confirmed using yeast transformants, and the experimental results showed that overexpression of the AtBAG2 sequence encoding only the BAG domain was sufficient to impart thermotolerance. Overall, these results suggest that the BAG domain-dependent molecular chaperone activity of AtBAG2 is indispensable for the heat stress response of Arabidopsis. This is the first report demonstrating the role of AtBAG2 as a sole molecular chaperone in Arabidopsis.


Introduction
B cell lymphoma 2 (Bcl2)-associated athanogene (BAG) family proteins are conserved across a wide range of eukaryotes including animals, yeast, and plants. BAG1, the first BAG protein to be discovered, was identified by screening for human Bcl-2-interacting proteins in a mouse embryo cDNA library [1][2][3]. To date, six BAG proteins (BAG1-BAG6), harboring a conserved BAG domain (BD) at the C-terminus have been reported in humans [4]. The three-dimensional structure of the BD, which contains approximately 110 amino acids (aa), comprises three α-helices, each containing 30-40 aa [5,6]. Through the BD, BAG proteins modulate the refolding activity of the heat shock-inducible heat shock protein 70 (HSP70) and the constitutively expressing heat shock cognate protein 70 (HSC70) by interacting with their ATPase domain [5]. In mammals, BAG proteins interact with members of diverse protein families to regulate various cellular phenomena such as protein degradation and cell apoptosis, migration, and proliferation [7,8].
In plants, BAG genes are associated with development and stress responses [9]. An advanced bioinformatics analysis revealed that the model plant Arabidopsis thaliana contains seven BAG proteins (AtBAG1-AtBAG7) [10,11]. Among these, AtBAG1-AtBAG4 carry an N-terminal ubiquitin-like (UBL) domain, whereas AtBAG5-AtBAG7 are plant-specific proteins harboring a calmodulin (CaM)-binding motif near the BD [10,11]. AtBAG proteins are reportedly involved in various processes related to plant development and environmental stress response. For example, AtBAG1 plays a critical role in the Hsc70-mediated proteasomal degradation of misfolded and unimported plastid proteins in the cytosol, and an optimal level of this protein is important for normal plant growth. AtBAG6, a CaM-binding protein, induces programmed cell death (PCD) in Arabidopsis and yeast, and its 134 aa stretch, which encompasses both the CaM-binding IQ motif and BD, is sufficient to induce cell death [11]. Additionally, AtBAG4, AtBAG6, and AtBAG7 are involved in the plant response to pathogen attack as well as the tolerance to cold, heat, ultraviolet (UV), and endoplasmic reticulum (ER) stresses [9,10,12]. Recently, AtBAG2 and AtBAG6 were reported to be involved in the tolerance to multiple abiotic stresses in Arabidopsis [13]. However, information on the biochemical and molecular functions of BAG proteins that affect plant physiology is extremely limited. In this study, we explored the intrinsic biochemical and molecular properties of AtBAG2 and characterized this protein as a novel molecular chaperone.

Plant Material and Growth Conditions
Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild type (WT) in this study. The homozygous T-DNA mutant atbag2 (SALK_030295; Col-0 background) was obtained from the Arabidopsis Biological Resource Center (ABRC; Ohio State University, Columbus, OH, USA). Seeds were sown in Petri dishes containing full-strength Murashige and Skoog (MS) medium (Duchefa Biochemie B.V., Haarlem, The Netherlands) supplemented with 2% (w/v) sucrose and 1% (w/v) agar, and cold-stratified in the dark at 4 • C for 3 days. The plates were then transferred to an environmentally controlled growth chamber maintained at 22 • C temperature, a 16 h light/8 h dark photoperiod, and 100 µmol m −2 s −1 light intensity. The chemicals were purchased from Sigma-Aldrich (Sigma-Aldrich Chemical, Saint Louis, MO, USA).

Heat Shock Treatment and Phenotypic Analysis of Plants
To examine the thermotolerance of the WT and atbag2 mutant, seedlings were grown on solid nutrient medium containing 2% (w/v) sucrose, and incubated under the abovementioned conditions [14]. Plates containing 7-day-old seedlings were sealed with plastic electrical tape and submerged for 30 min in a water bath maintained at 44 • C. The plates were then transferred to normal growth conditions, and the thermotolerance of seedlings was determined by measuring their survival rate. To measure electrolyte leakage, the seedlings were submerged in 5 mL of deionized water and placed on a shaker at 22 • C. The initial conductivity of the solution was measured using an Orion 3-Star Benchtop Conductivity Meter (Thermo Electron Cooperation, Rosemount, MN, USA). The seedlings were then autoclaved for 15 min, and conductivity was measured again (final conductivity) to determine the total amount of ions in solution. Percent ion leakage was estimated as the ratio of initial conductivity to final conductivity.
Samples of vector, G-AtBAG2, G-AtBAG2-Nt, G-AtBAG2-M, and G-AtBAG2-Ct transformants collected at the 60 min time point were subjected to trypan blue (TB) staining and spot assays. The TB staining assay was performed as described previously [16]. To conduct spot assays, the concentration of each culture was adjusted to an optical density (OD 600 ) of 1.0. Next, 6 µL aliquots of 10-fold serial dilutions were spotted onto YPD agar plates, incubated at 27 • C, and examined after 2-3 days.
The chaperone activity of recombinant proteins was measured using citrate synthase (CS), malate dehydrogenase (MDH), and insulin (Sigma-Aldrich) as substrates, as described previously [19,20]. Turbidity caused by substrate aggregation was monitored using the DU800 spectrophotometer (Beckman Coulter, Indianapolis, IN, USA) equipped with a thermostatic cell holder.

AtBAG2 Enhances Heat Shock Tolerance in Arabidopsis
To confirm the physiological significance of AtBAG2 in plant stress response, we selected homozygous lines of the atbag2 T-DNA insertion mutant originally obtained from ABRC (OH, USA) ( Figures 1A,B, S1 and S2). Transcript levels of AtBAG2 were compared between WT (Col-0) and atbag2 mutant plants by semi-quantitative reverse transcription PCR (sqRT-PCR) ( Figures 1C, S3 and S4). Although the AtBAG2 signal was clearly detected in WT plants, it was barely detectable in the atbag2 mutant, as expected ( Figures 1C and S3). By contrast, the signal for the Actin-2 (ACT2) gene (AT3G18780), which was used as a control, was nearly identical in both WT and atbag2 samples ( Figures 1C and S4). Next, we investigated the physiological function of AtBAG2 in 7-day-old WT and atbag2 seedlings under heat shock conditions ( Figure 1D-F). Under normal conditions (22 • C), the two genotypes exhibited no difference in seed germination and seedling survival rates ( Figure 1D,E). However, when incubated at 44 • C for 30 min, the 7-day-old atbag2 seedlings exhibited outstanding thermosensitivity ( Figure 1D,E), as indicated by their low survival rate (31.4 ± 5.6%) compared with the WT (56.6 ± 5.2%) ( Figure 1E). Because heat shock causes considerable damage to the channel and transporter proteins, fluidity, and other properties of the cell membrane, resulting in electrolyte leakage [21,22], we compared ion leakage between the WT and atbag2 plants. Following incubation at 44 • C for 30 min, the seedlings of both genotypes exhibited an increase in ion leakage; however, the level of ion leakage was higher in atbag2 seedlings than in the WT ( Figure 1F). The WT and atbag2 seedlings took 53 and 37 min, respectively, to reach 50% ion leakage. Overall, these results indicate that AtBAG2 plays a major role in plant survival under heat stress. and atbag2 seedlings grown at optimal temperature (22 • C) for 14 days (control) or subjected to heat stress conditions (7-day-old seedlings treated with heat shock [44 • C] for 30 min, and then grown at 22 • C for 7 days). (E,F) Comparison of survival rate (E) and ion leakage (F) between 7-day-old WT (Col-0) and atbag2 seedlings subjected to the heat shock treatment, as described in (D). In (E), the survival rates of heat-stressed Col-0 and atbag2 seedlings (heat shock) were compared with those of unstressed plants (control). Data represent the mean ± standard deviation (SD) of at least three independent experiments. To measure the ion leakage of Col-0 (-•-) and atbag2 (--) seedlings shown in (F), samples were collected at the indicated times and submerged in deionized water for 1 day. The conductivity of at least 10 seedlings was measured before autoclaving (initial conductivity) and after autoclaving (final conductivity). Data represent the mean ± SD of at least three independent experiments.

AtBAG2 Overexpression Enhances Thermotolerance in Yeast
Because atbag2 plants were more sensitive to heat shock than WT plants, we investigated whether AtBAG2 imparts heat stress tolerance at the cellular level by monitoring the effects of AtBAG2 overexpression in yeast cells. To conduct this experiment, we cloned the GST-AtBAG2 gene fusion under the control of the GAL1 promoter to generate the pYES2-G-AtBAG2 construct. We then transformed pYES2-G-AtBAG2 and the GST (G)-only construct (pYES2-G) separately into yeast W303 cells for the conditional overexpression of recombinant proteins. To confirm the physiological significance of AtBAG2 overexpression in yeast, we compared the viability of heat-shocked G and G-AtBAG2 transformants. Cultures of the two transformants were adjusted to equal cell densities at the mid-exponential growth stage, and aliquots were incubated at 27 • C or 55 • C. Viable cell counts were measured at regular intervals (Figure 2A). The Gand G-AtBAG2-transformed cells showed no significant difference in percent survival upon incubation at 27 • C for up to 60 min. However, at 55 • C, the survival rate of G transformants was dramatically lower than that of G-AtBAG2 transformants (57.6 ± 4.73% and 80.9 ± 3.97%, respectively, at 30 min; 43.3 ± 4.22% and 73.6 ± 5.37%, respectively, at 60 min). To confirm these results, we performed the TB exclusion assay [23]; in this assay, only dead cells are expected to stain blue as they cannot exclude the dye. No TB-positive Gand G-AtBAG2-transformed cells were observed at 27 • C after 60 min ( Figure 2B). However, after a 60 min incubation at 55 • C, more than 50% of the G-transformed cells appeared to be dead (intense blue staining), while the G-AtBAG2 transformants were alive (mild blue staining) ( Figure 2B). Taken together, our results consistently demonstrated that G-AtBAG2 overexpression resulted in a remarkable increase in the thermotolerance of yeast cells.

AtBAG2 Functions as a Molecular Chaperone
Since AtBAG2 conferred thermotolerance in both Arabidopsis and yeast, we explored the mechanism of action of the AtBAG2 protein by examining whether it exhibits molecular chaperone activity. To study the molecular chaperone activity of AtBAG2, we examined its surface hydrophobicity using bis-ANS, which binds to hydrophobic patches on proteins and fluoresces at an emission maximum of~470 nm [24], and explored its ability to prevent the heat-induced aggregation of CS and MDH or dithiothreitol (DTT)-induced aggregation of insulin by measuring light scattering at 340 nm. The hydrophobicity of GST-fused AtBAG2 (G-AtBAG2) and its ability to prevent protein aggregation were compared with those of GST-fused Ypt1p (G-Ypt1p), a previously characterized molecular chaperone [19] (positive control), and GST alone (G; negative control). The fluorescence signal of bis-ANS-bound G-AtBAG2 was greater than that of G-Ypt1p ( Figure 3A), suggesting that the chaperone activity of G-AtBAG2 is higher than that of G-Ypt1p. High surface hydrophobicity is a typical characteristic of molecular chaperones [25]. Based on these results, we investigated the chaperone activity of G-AtBAG2, along with those of G-Ypt1p (positive control) and G (negative control). In light scattering experiments, when CS (2 µM), a substrate protein [19,20], was heated alone or in the presence of 5-fold molar excess (8.35 µM) of the G protein, light scattering increased rapidly; however, when 2 µM CS was heated in the presence of 5-fold molar excess of G-AtBAG2 and G-Ypt1p, its aggregation was successively inhibited, confirming that G-AtBAG2 and G-Ypt1p function as molecular chaperones ( Figure 3B). Interestingly, the aggregation prevention activity of G-AtBAG2 was greater than that of G-Ypt1p ( Figure 3B). Furthermore, when MDH (1.67 µM) was heated alone or along with a two-fold molar excess (3.34 µM) of G, light scattering increased rapidly; however, when 1.67 µM MDH was heated in the presence of increasing amounts of G-AtBAG2 (0.5-, 1-, and 2-fold molar excess), its aggregation was inhibited in a concentration-dependent manner, confirming that G-AtBAG2 exhibits chaperone activity (Figure 3C). When G-AtBAG2 was present at two-fold molar excess (3.34 µM), approximately 90% of the MDH protein was protected against heat-induced aggregation during the 15 min incubation period ( Figure 3C). G-AtBAG2 also inhibited the DTT-induced aggregation of insulin in a concentration-dependent manner ( Figure 3D). These observations clearly demonstrate that AtBAG2 functions as a molecular chaperone.

BD Is Required for the Molecular Chaperone Activity of AtBAG2
The analysis of the protein domain structure on the InterPro website (http://www. ebi.ac.uk/interpro/) revealed that the amino acid sequence of AtBAG2 contains one UBL domain and one BD. Therefore, we investigated the roles of both of these domains in the molecular chaperone function of AtBAG2. First, we generated GST fusion constructs of three serial deletion derivatives of AtBAG2 (G-AtBAG2-Nt, G-AtBAG2-M, and G-AtBAG2-Ct) ( Figure 4A). Among the three AtBAG2 deletion variants, G-AtBAG2-Nt contains the UBL domain, G-AtBAG2-M contains the BD, and G-AtBAG2-Ct contains no unique domain. The recombinant GST fusion proteins were then produced in E. coli and purified, and their hydrophobicities were compared with that of G-AtBAG2 using bis-ANS. Upon binding to bis-ANS, G-AtBAG2-M showed high fluorescence, as with G-AtBAG2, whereas G-AtBAG2-Nt and G-AtBAG2-Ct showed very low fluorescence ( Figure 4B). We then compared the chaperone activities of G-AtBAG2-Nt, G-AtBAG2-M, and G-AtBAG2-Ct with that of G-AtBAG2 by measuring their ability to prevent the heat-induced denaturation of the substrate protein MDH. Light scattering increased rapidly when MDH (1.67 µM) was heated alone or with a 5-fold molar excess (8.35 µM) of G-AtBAG2-Nt and G-AtBAG2-Ct; however, when MDH (1.67 µM) was heated in the presence of a five-fold molar excess of G-AtBAG2 and G-AtBAG2-M, its aggregation was successively prevented (Figure 4C), confirming the molecular chaperone activity of G-AtBAG2-M and G-AtBAG2. Based on this information, we compared the effects of the overexpression of G-AtBAG2, G-AtBAG2-Nt, G-AtBAG2-M, and G-AtBAG2-Ct on the thermotolerance of yeast cells. The G-AtBAG2-Nt, G-AtBAG2-M, and G-AtBAG2-Ct genes were cloned under the control of the GAL1 promoter to conditionally overexpress the recombinant proteins, and the constructs were named as pYES2-G-AtBAG2-Nt, pYES2-G-AtBAG2-M, and pYES2-G-AtBAG2-Ct, respectively. These three constructs as well as pYES2-G-AtBAG2 and pYES2-G were separately transformed into the W303 cells. A spot assay was then performed using the heat-shocked G, G-AtBAG2, G-AtBAG2-Nt, G-AtBAG2-M, and G-AtBAG2-Ct transformants grown in media supplemented with 2% galactose (Gal) and sampled at the mid-exponential stage. The heat-shocked yeast cells overexpressing G, G-AtBAG2-Nt, and G-AtBAG2-Ct showed little difference in viability; by comparison, those overexpressing G-AtBAG2 and G-AtBAG2-M showed significantly greater viability ( Figure 4D). Taken together, these results suggest that the BD is critical for the molecular chaperone activity of AtBAG2, and overexpression of the BD-containing AtBAG2 gene sequence significantly improves the viability of yeast cells under heat stress.

Discussion
The BAG family proteins, known to act as co-chaperones, share a well-conserved BD at their C-terminus, which facilitates their interaction with HSP70/HSC70 [1,5]. For example, the most representative BAG protein, human BAG1, functions as a nucleotide exchange factor (NEF) for the chaperone HSP70, and triggers the release of chaperone-bound proteins as a co-chaperone [26,27]. AtBAG1 also interacts with HSC70 via the BD [3], thus acting as a cofactor of HSC70 for the proteasomal degradation of unimported plastid proteins. However, it is difficult to find literature on BAG protein activity. According to the results of this study, AtBAG2 performs its own biochemical function without associating with other proteins. In particular, AtBAG2 was found to function as a molecular chaperone that prevents the heat-or chemical-induced aggregation of substrate proteins. AtBAG2 exhibited superior molecular chaperone activity with CS, which has frequently been used as a general substrate in molecular chaperone studies compared with Ypt1 ( Figure 3B), which was previously identified as a molecular chaperone. In addition, AtBAG2 prevented the aggregation of thermally denatured MDH and chemically denatured insulin proteins, and its activity increased in a concentration-dependent manner ( Figure 3C,D). Molecular chaperones exhibit high surface hydrophobicity [19,28], and AtBAG2 was no exception ( Figure 3A). Based on these results, AtBAG2 was characterized as a novel molecular chaperone in this study.
To determine the region of AtBAG2 essential for its molecular chaperone activity, three deletion variants of the protein were generated, AtBAG2-Nt (UBL domain-containing N-terminal region), AtBAG2-Ct (domain-less C-terminal region), and AtBAG2-M (BDcontaining middle region), and the activity of each variant protein was tested. Among the three derivative proteins, only AtBAG2-M exhibited molecular chaperone activity ( Figure 4C), suggesting that the BD is responsible for the molecular chaperone function of AtBAG2. This suggests the possibility that the BD can act as a functionally active site in molecular chaperones, proposing new research directions and the applicability of BAG proteins in the future. In humans, the first and second helices of BAG1 BD are important for its binding to the serine/threonine kinase Raf-1 [29], while the second and third helices are known to mediate its binding to the ATPase domain of Hsc70/Hsp70 [30]. In the future, it will be interesting to find out which helices and amino acid residues in the BD of AtBAG2 are essential for the high hydrophobicity and molecular chaperone activity of the protein.
The maintenance of intracellular protein homeostasis is one of the most basic processes required for survival. Under stress conditions, intracellular proteins are often damaged and lose function, and molecular chaperones play a major role in maintaining life by preserving protein structure and function. For example, in humans, small heat shock proteins (sHsps), such as Hsp27, maintain cytoplasmic protein function and protect cells under stress conditions [31]. Ypt1p, a small GTPase in yeast, is generally responsible for intracellular trafficking; however, upon exposure to heat shock, Ypt1p undergoes structural changes which amplify its molecular chaperone function to protect cells [19]. In Arabidopsis, AtPP5 usually participates in cell signal transduction as a phosphatase; however, under thermal stress, it protects plants by acting as a molecular chaperone and preventing protein aggregation [32]. AtP3B, a ribosomal protein, exhibits high molecular chaperone activity, and its gene expression increases at high temperatures to protect Arabidopsis plants from heat-induced damage [28]. Similarly, AtBAG2 was found to exhibit excellent molecular chaperone activity in this study ( Figure 3). Moreover, the atbag2 mutant seedlings were sensitive to heat shock (Figure 1), whereas yeast cells overexpressing the AtBAG2 gene showed enhanced survival under heat stress conditions (Figure 2), indicating that AtBAG2 enhances thermotolerance. Unfortunately, AtBAG2 complementation lines in the atbag2 mutant background could not be obtained, and therefore phenotypically characterized, in this study. Given that AtBAG2 enhances the tolerance to multiple abiotic stresses by regulating the expression of downstream genes involved in abscisic acid (ABA) response and stress-induced reactive oxygen species (ROS) production [13], revealing the relationship between its stress response mechanism and molecular chaperone activity is a research task that has yet to be accomplished. Our results, which identified and characterized AtBAG2 as a novel molecular chaperone, are expected to expand the research horizon for key factors such as HSP70 that regulate essential cellular functions by associating with BAG proteins.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/life13030687/s1, Figure S1: The original image for the Figure 1B