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. 2019 Mar 20;180(2):1230–1240. doi: 10.1104/pp.18.01582

Inverse Correlation Between MPSR1 E3 Ubiquitin Ligase and HSP90.1 Balances Cytoplasmic Protein Quality Control1

Jong Hum Kim a,b,2, Tae Rin Oh a,b,2, Seok Keun Cho a,b, Seong Wook Yang a,b,c,3,4, Woo Taek Kim a,b,3,4
PMCID: PMC6548273  PMID: 30890661

The inverse correlation between MPSR1 and AtHSP90.1 via miR414 may adjust the set-point of the HSP90-mediated protein quality control process in response to increasing stress intensity in Arabidopsis.

Abstract

MISFOLDED PROTEIN SENSING RING1 (MPSR1) is a chaperone-independent E3 ubiquitin ligase that participates in protein quality control by eliminating misfolded proteins in Arabidopsis (Arabidopsis thaliana). Here, we report that in the early stages of proteotoxic stress, cellular levels of MPSR1 increased immediately, whereas levels of HEAT SHOCK PROTEIN90.1 (AtHSP90.1) were unaltered despite massively upregulated transcription. At this stage, the gene-silencing pathway mediated by microRNA 414 (miR414) suppressed AtHSP90.1 translation. By contrast, under prolonged stress, AtHSP90.1 was not suppressed, and instead competed with MPSR1 to act on misfolded proteins, promoting the destruction of MPSR1. Deficiency or excess of MPSR1 significantly abolished or intensified the suppression of AtHSP90.1, respectively. Similar to the MPSR1-overexpressing transgenic plants, the miR414-overexpressing plants showed an increased tolerance to proteotoxic stress as compared to the wild-type plants. Although the functional relationship between MPSR1 and miR414 remains unclear, both MPSR1 and miR414 demonstrated negative modulation of the expression of AtHSP90.1. The inverse correlation between MPSR1 and AtHSP90.1 via miR414 may adjust the set-point of the HSP90-mediated protein quality control process in response to increasing stress intensity in Arabidopsis.

Specific functions of cellular proteins are heavily dependent on their three-dimensional structure, which is achieved by a posttranslational folding process (Kim et al., 2013). The proper status of protein folding is critically affected by multiple intrinsic and extrinsic factors, including genetic mutations, proteotoxic chemicals, elevated temperature, and disrupted cellular osmotic and ionic homeostasis. To sustain cellular proteostasis against these detrimental stimuli, many molecular machineries, such as chaperones, the ubiquitin (Ub) 26S-proteasome system (UPS), proteinases, and autophagy, have developed in eukaryotic systems (Wickner et al., 1999; Kim et al., 2013; Hipp et al., 2014). In plants, protein-damaging stress is a secondary effect of many environmental stresses, including drought, high salinity, heat, and heavy metals, which hamper the growth and development of plants (Wang et al., 2004). To survive under those stresses, plants have evolved a large number of E3 Ub ligases that are critical in plant stress responses (Lee and Kim, 2011; Stone, 2014; Yu et al., 2016; Cho et al., 2017). Although 1,400 genes are known to encode E3 ligases in Arabidopsis (Arabidopsis thaliana; Vierstra, 2012), few E3 genes have been studied (Stone, 2014). In particular, the roles of cytoplasmic E3 Ub ligases in eliminating misfolded and damaged proteins are much less known. To date, only two cytoplasmic E3 ligases, CARBOXY TERMINUS OF HSC70 INTERACTING PROTEIN (AtCHIP) and MISFOLDED PROTEIN SENSING RING1 (MPSR1), have been characterized in Arabidopsis (Yan et al., 2003; Kim et al., 2017). AtCHIP directly interacts with HEAT SHOCK PROTEIN70 (HSP70) and tethers poly-Ubs to misfolded or damaged proteins. AtCHIP expressions are related to stress conditions including cold temperature, heat, and salt (Yan et al., 2003). Given the highly conserved HSP70 chaperone activity for a plethora of unfolded and damaged proteins (Mayer and Bukau, 2005), the complex of AtCHIP and HSP70 chaperone is a noteworthy case that plays a role in the protein quality control (PQC) pathway in plants (Qian et al., 2006).

Recently, we reported a cytosolic E3 Ub ligase MPSR1, which is critical in the response to protein-damaging stress in Arabidopsis (Kim et al., 2017). MPSR1 is a self-regulatory E3 Ub ligase; without proteotoxic stress, it is self-ubiquitinated and rapidly degraded by the 26S-proteasome, thereby concealing its PQC activity. Upon stress, MPSR1 is stabilized by misfolded client proteins and ubiquitinates them for degradation, independently of chaperones. Furthermore, MPSR1 excessiveness maintains plant growth under proteotoxic stress, possibly by attuning the stress threshold of the 26S-proteasome (Kim et al., 2017). However, chaperones generally dictate the balance among protein folding, degradation, and aggregation in eukaryotes (Morán Luengo et al., 2019), and therefore, to clearly understand the roles of MPSR1 in the cytoplasmic PQC in plants, temporal and spatial correlations between MPSR1 and cytoplasmic chaperones should be comprehended.

AtHSP90 and AtHSP70 are well-characterized molecular chaperones that are synthesized de novo under protein-damaging conditions in Arabidopsis (Wang et al., 2004). Seven members of the AtHSP90 gene family encode highly conserved 90-kD HSPs. Four of them are AtHSP90.1, AtHSP90.2, AtHSP90.3, and AtHSP90.4 that encode HSP90s, which are mainly localized in the cytoplasm. The other three 90-kD HSPs, namely AtHSP90.5, AtHSP90.6, and AtHSP90.7, are found in different organelles. Among the cytoplasmic AtHSP90 genes, only AtHSP90.1 is promptly induced by heat shock stress, whereas the other three are constitutively expressed (Yabe et al., 1994). Eighteen genes encode 70-kD HSPs in Arabidopsis. This HSP family is classified into two subfamilies: DnaK and HSP110/SSE (Stress Seventy family E; Usman et al., 2017). Among them, AtHSP70.1 and AtHSP70.2 are major isoforms in the cytoplasm (Usman et al., 2017). Thus, we investigated whether the functionality of MPSR1 as a frontline surveillance system is negatively or positively correlated with the two representative cytoplasmic chaperones AtHSP90.1 and AtHSP70.1 in Arabidopsis.

RESULTS

Inversely Correlated Expressions of MPSR1 and AtHSP90.1

To understand the interconnections between MPSR1 and cytoplasmic AtHSP90.1 and AtHSP70, we compared the accumulation profiles of MPSR1 to those of AHSP90.1 and AtHSP70 induced by azetidine-2-carboxylic acid (AZC), an analog of l-Pro that causes irreversible protein misfolding. While investigating each protein level of MPSR1, AtHSP90.1, and AtHSP70 by time-dependent AZC treatments, we found that MPSR1 accumulated earlier than AtHSP90.1 and AtHSP70. After 0.5–1 h of stress treatments, MPSR1 began to accumulate without a distinctive increase in the MPSR1 transcription (Fig. 1, A and B). Notably, protein levels of both AtHSP90.1 and AtHSP70 did not show any detectable changes at the initial stage (0.5–1 h) of stress (Fig. 1, A and B). After 2 h of AZC treatment, MPSR1 protein and transcript levels increased 4.5- and 4-fold, respectively. In contrast, the levels of AtHSP90.1 and AtHSP70 proteins remained unchanged (<1.2-fold), even though their transcripts were markedly elevated at ∼160-fold and 16-fold, respectively (Fig. 1B). After 3 h of AZC treatment, MPSR1 transcript reached its maximum level (∼ 8-fold induction), while AtHSP90.1 and AtHSP70.1 transcripts increased ∼180- and ∼20 times, respectively (Fig. 1, C and D). Noteworthy amounts of AtHSP90.1, and to a lesser extent AtHSP70, began to increase 3 h after AZC treatment. We also tested the transcript level of an E3 ligase AtCHIP in response to AZC; AtCHIP is a well-defined AtHSP90-dependent cytoplasmic PQC E3 Ub ligase. AtCHIP gradually increased up to 5-fold after 36 h (Fig. 1C), suggesting that it may have a relatively minor role in the early stage of the PQC pathway. When the stress was prolonged, the levels of AtHSP90.1 and AtHSP70 proteins further increased and reached their highest value at 24–36 h, with the induction of AtHSP90.1 being more evident than that of AtHSP70 (Fig. 1A). On the contrary, after its maximum level at 2 h, the MPSR1 protein level gradually declined thereafter to the level of nonstress condition at 36 h. Although AtHSP90.1 shares relatively lower amino acid sequence similarity with the other AtHSP90 proteins (Cha et al., 2013), we could not rule out the cross-activity of α-AtHSP90.1 antibody against the other subtypes. Using a commercially available antibody, we tested the expression pattern of AtHSP90.2. Interestingly, we found that AtHSP90.2 was slightly upregulated at the initial stage of stress (0.5 to 1 h) and then fluctuated in length of time (Supplemental Fig. S1). This expression pattern was clearly different from that of AtHSP90.1, showing the specificity of α-AtHSP90.1 antibody. Taken together, our results indicated that the expression of MPSR1 is inversely correlated to that of AtHSP90.1.

Figure 1.

Figure 1.

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Expression patterns of MPSR1 and AtHSP90.1. A, Time-dependent induction profiles of MPSR1, AtHSP90.1, and AtHSP70 proteins in response to AZC treatments. Wild-type (WT) Arabidopsis seedlings were incubated with 5 mM of AZC for 0–36 h. B, Accumulation patterns of MPSR1, AtHSP90.1, and AtHSP70 proteins and their transcripts in the early stages of proteotoxic stress (5 mM of AZC treatments for 0, 0.5, 1, and 2 h). Left: The relative level of each protein was calculated by image analysis and plotted in arbitrary units versus the level of the initial samples. Right: Relative amounts of MPSR1, AtHSP90.1, and AtHSP70.1 transcripts were determined by RT-qPCR. Error bars = means ± sd (n = 4) from four biological replicates. Red asterisks denote the time point at which the HSP90.1 protein level was unaltered despite its transcript level increasing more than 30-fold. C and D, Time-dependent expression profiles of MPSR1, AtCHIP, AtHSP90.1, and AtHSP70.1 transcripts induced by AZC (5 mM, 0–36 h) were examined by RT-qPCR. Error bars = means ± sd (n = 4) from four biological replicates.

AtHSP90.1 Facilitates MPSR1 Turnover by Competing Misfolded Substrate Proteins

Several studies reported that, in the triage of misfolded proteins, HSP90 either sequesters or guides its clients to chaperone-dependent E3 Ub ligases for degradation, whereas HSP70 mostly guides its clients for degradation in yeasts and mammals (Pratt et al., 2010, 2015). Therefore, we hypothesized that if AtHSP90.1 excessively accumulates enough to sequester most of the misfolded proteins, it could promote MPSR1 destabilization by self-ubiquitination. We tested this hypothesis using a transgenic Arabidopsis plant harboring the XVE:AtHSP90.1 expression construct. The transgenic seedlings were incubated with β-estradiol (20 μM) to excessively induce AtHSP90.1 or with a mock for the noninduced control (Fig. 2A). After 24 h of incubation, the XVE:AtHSP90.1 seedlings were additionally treated with AZC (5 mM) for 1 to 4 h. The level of MPSR1 protein was reduced by ∼5 times in the β-estradiol-treated seedlings relative to that in the mock-treated control without AZC and further reduced by ∼10 times in the presence of AZC (Fig. 2B). These results indicated that excess AtHSP90.1 decreased the level of MPSR1 protein. The transcript of MPSR1 was rather increased in the β-estradiol–pretreated samples, confirming the proteolytic reduction of MPSR1 in AtHSP90.1-overexpressors (OEs; Fig. 2C).

Figure 2.

Figure 2.

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AtHSP90.1 facilitates MPSR1 turnover by competing for misfolded substrate proteins. A, Relative expression levels of AtHSP90.1 transcripts in XVE:AtHSP90.1 transgenic plants. Transgenic plants were treated with β-estradiol and AZC. B, Accumulation of MPSR1 under AZC treatment was suppressed by β-estradiol–induced AtHSP90.1. XVE:AtHSP90.1 transgenic seedlings were incubated with or without β-estradiol (20 μM) for 24 h to induce AtHSP90.1 and subsequently treated with AZC (5 mM) for 1–4 h. Levels of MPSR1 and AtHSP90.1 proteins were determined with α-MPSR1 and α-AtHSP90.1 antibodies, respectively. Histone H3 was used as an equal loading control. C, Relative expression levels of MPSR1 transcripts in the XVE:AtHSP90.1 plants. Transgenic plants were treated with β-estradiol and AZC as described in (B). D, In vitro competition pull-down assay. Bacterially expressed GST-MPSR1 and His-Δ2GFP were incubated with or without different amounts of MBP-AtHSP90.1 in the presence of a Ni-NTA resin. Eluted proteins were separated by SDS-PAGE and detected by immunoblotting using α-GST, α-HSP90, and α-GFP antibodies. E, AtHSP90.1 restores self-ubiquitinating E3 ligase activity of MPSR1 by sequestering misfolded Δ2GFP. GST-MPSR1 was subjected to an in vitro self-ubiquitination assay in the presence or absence of Ub, AtUBA1 (E1), AtUBC8 (E2), His-Δ2GFP, and MBP-AtHSP90.1 as indicated. Reaction mixtures were resolved by SDS-PAGE and analyzed by immunoblotting with α-GST antibody. Red square brackets indicate ubiquitinated proteins. Blue arrowheads indicate unmodified GST-MPSR1. Error bars in (A) and (C) = means ± sd (n = 4) from four biological replicates.

To investigate whether AtHSP90.1 suppresses MPSR1 by competing for misfolded client proteins, we performed a competition pull-down assay with Δ2GFP, an artificial misfolded protein; previously, we showed that Δ2GFP with a deletion of the 13N-terminal amino acids is an effective mimicry of misfolded proteins (Kim et al., 2017). Interestingly, we observed that the interaction between glutathione (GST)-MPSR1 and histidine (His)-Δ2GFP was hindered by maltose-binding protein (MBP)-AtHSP90.1 in a concentration-dependent manner (Fig. 2D). Furthermore, using the in vitro ubiquitination assay, we confirmed the notion that AtHSP90.1 competes with MPSR1 for misfolded proteins. In the presence of His-Δ2GFP, self-ubiquitinating E3 ligase activity of GST-MPSR1 was markedly reduced (Fig. 2E). However, addition of MBP-AtHSP90.1 in the reaction mixtures restored the self-ubiquitination of GST-MPSR1 (Fig. 2E). In contrast, addition of MBP failed to restore the self-ubiquitinating activity of GST-MPSR1 (Supplemental Fig. S2). These results indicated that AtHSP90.1 negatively regulates MPSR1 activity by competitively sequestering misfolded Δ2GFP proteins. Hence, under the prolonged stress condition, the dramatically accumulated AtHSP90.1 could promote the destruction of MPSR1 by the 26S-proteasome (Fig. 1A).

Expression of AtHSP90.1 Is Regulated by microRNA 414-Mediated Gene Silencing

The discordance between transcription and translation of AHSP90.1 at the initial stage of stress (Fig. 1A) prompted us to seek the molecular mechanism underlying the phenomenon. We formulated two plausible scenarios to explain the mechanism, by which the expressions of MPSR1 and AtHSP90.1 are differently regulated in an opposing fashion by proteotoxic stress. The first scenario was that AtHSP90.1 is degraded in a posttranslational manner at the early stage of stress. To test this hypothesis, we examined the AtHSP90.1 protein levels in the wild-type seedlings treated with AZC or with AZC plus MG132 and protease inhibitor cocktail (PI). The former is a well-known proteasome inhibitor and the latter is a mixture of inhibitors for proteases and autophagy pathway. Immunoblot results revealed that there is little difference in the amount of AtHSP90.1 in the two different samples, indicating that AtHSP90.1 is stable at the early stage of stress (Supplemental Fig. S3). Thus, the first scenario, the posttranslational turnover of AtHSP90.1, was unlikely.

Alternatively, the second scenario was the translational suppression of AtHSP90.1 by micro-RNA (miRNA)-mediated gene silencing. We found that only AtHSP90.1 has a complementary sequence to microRNA 414 (miR414) among the four subtypes (Fig. 3A; Supplemental Fig. S4). Therefore, we examined the levels of AtHSP90.1 protein in the miRNA-deficient mutant lines such as hyl1-2 and hen1-1. The former is deficient in HYPONASTIC LEAVES1 (HYL1), which encodes a core miRNA binding protein, and the latter is defected in the HUA ENHANCER1 (HEN1) gene for miRNA methyltransferase. AtHSP90.1 highly accumulated in hyl1-2 and hen1-1 mutant lines as compared to control at the early stage of proteotoxic stress (Fig. 3B). ARGONAUTE1 (AGO1) is a crucial component of RNA-induced silencing complex that cleaves target mRNAs or suppresses the translation of target mRNAs (Baumberger and Baulcombe, 2005). Therefore, we monitored the level of AtHSP90.1 in the ago1-27 mutant line. As seen in the hyl-1 and hen1-1 mutant lines, the amount of AtHSP90.1 was higher in the ago1-27 mutant than in the wild-type plants (Supplemental Fig. S5). The level of AtHSP90.1 transcript was scarcely different in each genotype, supporting the second hypothesis (Fig. 3C). However, it might be argued that the accumulations of AtHSP90.1 in hyl1-2, hen1-1, and ago1-27 were caused by the pleiotropic defects in the miRNA-deficient mutants. To rule out this possibility, we constructed 35S:miR414-OE transgenic lines (Supplemental Fig. S6), which showed no defective phenotypes under normal conditions. Then, we observed that the levels of AtHSP90.1 protein were notably reduced in the 35S:miR414 transgenic lines under the AZC-treated condition, but there was no significant change in AtHSP90.1 transcript level, implying that the translational suppression of AtHSP90.1 was at the early stage of proteotoxic stress (Fig. 3D and Supplemental Fig. S7). When the stress maintained over 3 h, the suppression of AtHSP90.1 was lifted in the wild-type seedlings, possibly because of the gradual reduction of endogenous miR414 (Fig. 3E). The reduction of miR414 was inversely correlated with the increased levels of primary (pri)-miR414 transcripts, implying that the AZC treatment can cause a possible hindrance in the processing of miR414 (Fig. 3F).

Figure 3.

Figure 3.

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Negative regulation of AtHSP90.1 by miR414. A, Predicted miRNA414 target site in the AtHSP90.1 transcript. B and C, Expression levels of AtHSP90.1 protein (B) and transcript (C) in the AZC-treated wild-type and miRNA-deficient mutants, hyl1-2 and hen1-1, were detected by immunoblot and RT-qPCR analyses, respectively. Error bars in (C) = means ± sd (n = 3) from three biological replicates. D, Expression levels of AtHSP90.1 protein in AZC-treated wild-type and miR414-OE transgenic plants. E, Expression levels of miR414 in mock and AZC-treated wild-type plants. 5S rRNA was used as a loading control. F, Results of RT-qPCR analysis of pri-miR414 in wild-type plants under AZC-treated conditions. Error bars in (F) = means ± sd (n = 4) from four biological replicates. WT, wild type.

Because miRNAs recognize multiple targets (Zhang et al., 2010), the 35S:miR414 transgenic line intrinsically harbors a possibility of extra target silencing, which could affect plant physiology regardless of the stress treatments. To foreclose the possibility, we additionally constructed XVE:miR414 transgenic lines, in which the expression of miR414 can be chemically controlled by an inducible XVE promoter system. By adding β-estradiol (20 μM), we induced the expression of miR414 for 24 h before treating proteotoxic stress (Fig. 4A). The accumulation of AtHSP90.1 was lowered by miR414 expression, confirming the miR414-mediated suppression of AtHSP90.1 translation (Fig. 4A). Consistent with these notions, the 35S:miR414 seedlings displayed markedly increased tolerant phenotypes in response to AZC treatments as compared to the wild-type plants in both germination and postgermination stages (Fig. 4, B–D). These tolerant phenotypes of 35S:miR414 plants were reminiscent of those of 35S:MPSR1 lines (Kim et al., 2017), further indicating a positive correlation between miR414 and MPSR1.

Figure 4.

Figure 4.

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Overexpression of miR414 results in improved tolerance against proteotoxic stress. A, Protein levels of AtHSP90.1 in AZC-treated wild-type (WT) and XVE:miR414 transgenic plants (independent lines #1 and #3). Before AZC treatment, miR414 was induced through β-estradiol treatment. Histone H3 was used as a loading control. B and C, AZC-tolerant phenotype of 35S:miR414-OE transgenic plants in the germination stage. Cotyledon greening percentages were determined 7 d after germination. Results are presented as the means ± sd (**P < 0.01, Student’s t test) of three independent biological replicates (n = 25). D, AZC-tolerant phenotype of 35S:miR414-OE transgenic plants in the postgermination stage. Scale bars = 5 mm.

MPSR1 Suppresses the Expression of AtHSP90.1 Protein

We also noticed that the accumulation of AtHSP90.1 could be suppressed or delayed by MPSR1 at the early stage of proteotoxic stress. Indeed, higher amounts (approximately sixfold) of AtHSP90.1 were detected in the 35S:MPSR1-knockdown (KD) transgenic lines in response to AZC treatment (5 mM for 3 h) as compared to those in the wild-type plants, indicating that the MPSR1-deficiency resulted in the increased accumulation of AtHSP90.1 (Fig. 5A). As negative controls, LUMINAL BINDING PROTEIN 2 (Bip2) and calnexin, both of which are endoplasmic reticulum-associated chaperones, were tested. The results showed that amounts of these proteins were consistent in the wild-type and 35S:MPSR1-KD plants (Supplemental Fig. S8). In contrast, overexpression of MPSR1 reduced the level of AtHSP90.1. After 6 h of AZC treatment, the AtHSP90.1 level was distinctively reduced to ∼30% in the 35S:MPSR1-OE progeny relative to that of the wild-type (Fig. 5B). These results suggest the inverse correlation between MPSR1 and AtHSP90.1 in response to AZC. To find clues about the regulation of the AtHSP90.1 protein level by MPSR1, we next checked the transcript levels of AtHSP90.1 in the wild-type, 35S:MPSR1-KD, and 35S:MPSR1-OE plants in the presence of AZC (5 mM) for 3–6 h. There was no significant change in the transcript levels of AtHSP90.1 in the wild-type, 35S:MPSR1-KD, or 35S:MPSR1-OE plants, contrary to their protein levels (Fig. 5C). We further tested the MPSR1-mediated suppression of AtHSP90.1 in a time-dependent manner. After 20 h of stress treatment, the expression of AtHSP90.1 was upregulated in the 35S:MPSR1-KD plants (fold increase 4.0 versus 7.8), whereas it was suppressed in the 35S:MPSR1-OE plants (fold increase 4.8 versus 2.9), confirming the negative role of MPSR1 in AtHSP90.1 expression (Fig. 5, D and E). In fact, MPSR1 does not directly interact with AtHSP90.1 (Kim et al., 2017). Therefore, we suggest that the suppression of AtHSP90.1 could be indirectly regulated by MPSR1.

Figure 5.

Figure 5.

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MPSR1 suppresses the protein level of AtHSP90.1. A and B, Protein level of AtHSP90.1 is increased in 35S:MPSR1-KD (A) and decreased in 35S:MPSR1-OE (B) in response to AZC. Wild-type, 35S:MPSR1-KD (independent lines #1 and #2), and 35S:MPSR1-OE (independent lines #2 and #9) plants were incubated with 5 mM of AZC for 3 h and 6 h, respectively. Level of AtHSP90.1 was detected by immunoblotting with α-AtHSP90.1 antibody. Histone H3 was used as a loading control. C, Transcript levels of AtHSP90.1 in wild type, 35S:MPSR1-KD (lines #1 and #2), and 35S:MPSR1-OE (lines #2 and #9) plants. The amount of AtHSP90.1 mRNA was determined by RT-qPCR. Data are the average values of three biological replicates ± sd (n = 3). D and E, Time-course expression levels of AtHSP90.1 in 35S:MPSR1-KD (D) and 35S:MPSR1-OE (E) transgenic plants as compared with wild-type plants under AZC-treated conditions. Histone H3 was used as a loading control. WT, wild type.

MPSR1 Excessiveness Specifically Alters the Transcriptome in Response to Proteotoxic Stress

Because MPSR1 excessiveness notably alleviates proteotoxic stress on plant growth (Kim et al., 2017), we tested whether the overexpression of MPSR1 influences the stress-responsive transcriptome. After 6 h of AZC treatment, 1,707 of mRNAs (change ratio: log2Δ fragments per kilobase million value > 0.5) showed increased expression in the wild-type seedlings out of the 17,798 mRNAs that had total expression of at least 10 fragments per kilobase million, while 1,417 of mRNAs increased in MPSR1-OE transgenic seedlings (Fig. 6A). By contrast, 2,193 genes and 1,809 genes decreased in the wild-type and the MPSR1-OE transgenic seedlings, respectively (Fig. 6A). Within the fluctuated genes, we further analyzed the differentially expressed genes in the MPSR1-OE seedlings as compared to those in the wild-type. The LATE EMBRYOGENIC ABUNDANT proteins protect other proteins from aggregation, which is caused by environmental stresses (Amara et al., 2013, 2014; Liang et al., 2013). The stress-induced transcription levels of LATE EMBRYOGENIC ABUNDANT family genes were distinctively lower in the MPSR1-OE transgenic seedlings than in the wild-type seedlings (Fig. 6B). Likewise, two positive regulatory genes in abiotic stress response, ZINC TRANSPORTER OF ARABIDOPSIS THALIANA18 and TOLERANT TO CHILLING AND FREEZING1, were much less expressed in the MPSR1-OEs than in the wild-type (Fig. 6B). The former is a Cys-2/His-2 zinc finger transcription factor that plays an essential role in response to various abiotic stresses, and the latter is a histone-binding protein that regulates freezing tolerance through modulating lignin biosynthesis (Ji et al., 2015; Yin et al., 2017). CALMODULIN LIKE39, which encodes a protein for plant development and stress responses (Bender et al., 2013; Midhat et al., 2018), was notably reduced in the MPSR1-OE transgenic seedlings (Fig. 6B). These results implied that MPSR1 excessiveness mitigated a certain degree of the proteotoxic stress and lowered the level of stress-responsive gene expressions. However, as compared to the wild-type seedlings, many chaperones and heat shock factor genes were unaltered or slightly reduced in the MPSR1-OE transgenic seedlings (Supplemental Fig. S9). We speculated that the transcriptional regulation of chaperones and heat shock factors might be not related to the functions of MPSR1. On the other hand, expressions of many genes for plant growth and developments—such as AUXIN UP-REGULATED F-BOX PROTEIN2, which regulates auxin transport and cytokinin signaling for root growth (Zheng et al., 2011), and ROOT MERISTEM GROWTH FACTOR8 gene, which is involved in root cell maintenance and cell proliferation (Matsuzaki et al., 2010; Fernandez et al., 2015)—maintained in the MPSR1-OE transgenic seedlings (Fig. 6C). Contrastively, expressions of these genes were notably dropped in the wild-type seedlings (Fig. 6C). These results suggested that the MPSR1 excessiveness might allay plant proteotoxic stress responses, thereby balancing the transcriptome shift toward maintaining growth. Moreover, to find a clue for the relation between MPSR1 and miR414, we analyzed the transcription levels of miRNA-biogenetic component genes, including DCL1-Like protein, SERRATE, HYL1, AGO1, and HEN1. Under the stress condition, the MPSR1 excessiveness did not distinctively influence the expressions of DCL1-Like protein, HYL1, SERRATE, and AGO1. Only HEN1 slightly decreased in the MPSR1-OE transgenic seedlings (Fig. 6D). However, this analysis was insufficient to explain the stress-dependent reduction of miR414 production (Fig. 3E) and a possible role of MPSR1 in miR414 biogenesis.

Figure 6.

Figure 6.

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Excess MPSR1 alters the expressions of several stress-responsive genes. A, Heat-map of total mRNA level (fold-change) in wild type and 35S:MPSR1-OE by 6 h of 0.5 mM of AZC treatment. B–D, Heat-maps and graphs representing the expression levels of upregulated genes (B), downregulated genes (C), and miRNA biogenetic component genes (D) in wild type and MPSR1-OE by 6 h of 0.5 mM AZC treatment. E, Working model of the MPSR1-miR414-AtHSP90.1 triangular regulatory circuit in response to proteotoxic stress. In the early stages of proteotoxic stress, (i) MPSR1 might be positively associated with miR414 function and (ii) miR414 inhibits AtHSP90.1 by translational suppression. When stress is prolonged, (iii) AtHSP90.1 escapes from miR414-mediated suppression and binds to misfolded proteins, competing with MPSR1. This results in MPSR1 self-ubiquitination and degradation. WT, wild type.

DISCUSSION

Multifaceted mechanisms to monitor and resolve the challenges from damaged and unhealthy proteins are essential to maintain an appropriate cellular proteostasis (Kim et al., 2013; Hipp et al., 2014; Wolff et al., 2014). Two prominent modulators of cellular proteostasis are molecular chaperones and UPS in eukaryotes. Five major families of chaperones—HSP70s, HSP90s, chaperonins, HSP100s, and small HSPs—are expressed in multiple cellular compartments to engage a diverse misfolded and damaged proteins (Wang et al., 2004; Cha et al., 2013; Usman et al., 2017). Individual members of each class of the chaperones have particular roles, and their cooperative networks with themselves and UPS are seemingly a central principle of the integrated chaperone machinery in yeast and animals (Shiber and Ravid, 2014; Fernández-Fernández et al., 2017; Morán Luengo et al., 2019). In plants, several studies reported that cooperative networks of chaperones and UPS play roles in stress responses. For example, AtCHIP is involved in heat stress tolerance and abscisic acid responses (Yan et al., 2003; Luo et al., 2006; Zhou et al., 2014). AtCHIP and HSP70 remove the plastid-targeted proteins that can form nonspecific aggregations in the cytoplasm (Lee et al., 2009). In the case of the HSP90 family, two cytoplasmic AtHSP90.2 and AtHSP90.3 form a complex with Suppressor of the G2 Allele of Skp1, a cochaperone that functions in the formation of Skp1–Cul1–F-box protein E3 Ub ligase complex, to degrade immune receptors (Huang et al., 2014). Although the subtype was not clearly defined, Wang et al. (2016) showed that HSP90s and Suppressor of the G2 Allele of Skp1 also integrate temperature and auxin signaling and regulate plant growth in changing temperature. A substrate receptor of Cullin4-RING E3 ligase, HEAT STRESS TOLERANT DWD1, seems to be negatively associated with AtHSP90.1 (Kim et al., 2014). Furthermore, ZEITLUPE E3 ligase, a central circadian clock component, eliminates aggregated proteins through direct association with AtHSP90.1 (Gil et al., 2017).

In addition, we here showed that the expression of AtHSP90.1 is inversely correlated to that of MPSR1, a solo-playing PQC E3 Ub ligase (Kim et al., 2017). At the early stage of stress, MPSR1, as a fast-responding PQC E3 ligase, eliminates misfolded and damaged proteins, and possibly adjusts the levels of AtHSP90.1. Furthermore, we showed that the posttranscriptional regulation possibly suppresses AtHSP90.1 through the miR414-mediated gene silencing (Fig. 3). Notably, overexpression of either MPSR1 or miR414 hinders the expression of AtHSP90.1, which seems to be important in maintaining plant growth under the stress condition (Kim et al., 2017; Fig. 4). This process might be essential for plants in sensing the intensity of stress, by which plants decide the maintenance or pause of growth (Fig. 6, A–D). On the contrary, AtHSP90.1 suppresses the expression of MPSR1 by competing for aberrant proteins, showing the role of HSP90.1 in PQC E3 ligase activity under prolonged stress (Fig. 2).

Overall, our finding suggests two notions: The inverse correlation between MPSR1 and AtHSP90.1 via miR414 can be essential to modulate the HSP90.1-mediated PQC pathway in response to increasing stress intensity; and AtHSP90.1 is posttranscriptionally regulated in plants (Fig. 6E). However, many questions about the coordinated networks between AtHSP90s and UPS have still to be answered in plants: How does each isoform in the cytoplasmic HSP90 family differently recognize and determine the fate of its substrates? Which isoform stabilizes or protects from aggregations, facilitates refolding, and eventually eliminates substrates via the UPS-mediated degradation or autophagy pathway? Is there a central determinant that coordinates the HSP90 family-UPS network? To answer these questions, as the next challenge we will define how MPSR1 positively regulates the expression of miR414 and/or other molecules and therefore suppresses AtHSP90.1 at the early stage of proteotoxic stress.

MATERIALS AND METHODS

Constructions, Plant Materials, and Growth Conditions

Wild-type Arabidopsis (Arabidopsis thaliana) ecotype Col-0 (CS70000) and transgenic plants were grown on 1× Murashige and Skoog medium supplemented with 1% (w/v) Suc and 0.8% (w/v) phytoagar (Duchefa Biochemie) in a growth chamber (Panasonic) at 22°C under long-day conditions (16-h light/8-h dark) and a growth room at 22°C under constant light as described by Kim and Kim (2013). To generate 35S:miR414, pri-miRNA of the miR414 gene was cloned to pEG100 vector. To generate the XVE:AtHSP90.1 and XVE:miR414 transgenic plants, the full-length complementary DNA (cDNA) of AtHSP90.1 and genomic DNA of miR414 gene were cloned into the pMDC7 vector. The constructed plasmids were transformed into Arabidopsis plants using the method described by Ryu et al. (2010) using Agrobacterium tumefaciens (strain GV3101). Transgenic plants were selected on the growth medium supplemented with hygromycin (35 μg/mL; Duchefa) or glufosinate (25 μg/mL; Duchefa) and further grown in a growth room as described by Cho et al. (2011). To induce β-estradiol–inducible transgenes, 10-d–old transgenic seedlings were transferred to the liquid Murashige and Skoog medium supplemented with 20 μM of β-estradiol (Sigma-Aldrich) and incubated for 4–8 h or 24 h. Primers used for cloning are listed in Supplemental Table S1.

Immunoblot Analysis

Immunoblot analysis was conducted as described by Oh et al. (2017). Briefly, 10-d–old seedlings were ground in liquid N using a mortar and pestle. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The proteins on the blots were detected using α-AtHSP90.1 (1:10,000 dilution; Agrisera), α-AtHSP70 (1:5,000 dilution; Agrisera; α-AtHSP70 was generated with the peptide immunogen, which is conserved among three cytosolic HSP70, variants HSP70 [HSC70-4, AT3g12580], HSP70-1 [HSC70-1, AT5G02500], and HSP70B [At1g16030]), α-GST (1:5,000 dilution; Santa Cruz Biotechnology), α-MBP (1:5,000 dilution; ABM), α-MPSR1 (1:2,000 dilution; Abmart), α-Actin (1:10,000 dilution; Agrisera), α-Histone H3 (1:10,000 dilution; Agrisera), α-GFP (1:5,000 dilution; Clontech), α-calnexin (1:3,000 dilution; Agrisera), α-Bip2 (1:3,000 dilution; Agrisera), α-mouse (1:10,000 dilution; Amersham), and α-rabbit (1:10,000 dilution; Amersham) antibodies. The immunoblot band signals were quantified using the software ImageJ (National Institutes of Health).

Real-Time PCR, Real-Time Quantitative PCR, and Two-Tailed Real-Time PCR for Analyzing miRNA

For real-time PCR (RT-PCR) and real-time quantitative PCR (RT-qPCR), total RNA was extracted from 10-d–old Arabidopsis seedlings using a Plant Total RNA Extraction Kit (Intron) according to the manufacturer’s protocol. Total RNA (1–2 μg) was used for cDNA synthesis using the cDNA Synthesis Kit (Intron). The 1/10 cDNA was amplified by PCR using iTaq polymerase (Intron) with gene-specific–primer sets for AtHSP90.1, pri-miR414, and UBC10. RT-qPCR was performed using a PikoReal RT-PCR System (Thermo Fisher Scientific), as described by Seo et al. (2012). The relative amounts of MPSR1, AtHSP90.1, AtHSP70.1, and AtCHIP transcripts were measured by calibrating the threshold cycles of the target genes with 18S rRNA threshold cycles. Calculations were performed using the equation 2−ΔΔCT, where CT is the cycle number at which the fluorescence reaches the threshold point for detection. The experiments were performed with four independent biological replicates. The gene-specific primers used for RT-qPCR are listed in Supplemental Table S1.

For analyzing the levels of miRNA, we used the two-tailed RT-PCR method with simple modification (Androvic et al., 2017). Total RNA of 10-d–old Arabidopsis seedlings was isolated using a miVana miRNA Isolation Kit (Invitrogen) and 3–4 μg of total RNA was used for cDNA synthesis using a cDNA Synthesis Kit (Intron). Specificity of amplified miRNA414 amplicon was confirmed using an experimental set without RNA as a negative control. Information on primers used is in Supplemental Table S1.

Recombinant Protein Preparation

Purification of bacterially expressed GST-MPSR1 and His-Δ2GFP recombinant proteins was performed as described by Seo et al. (2016). The full-length coding sequence of AtHSP90.1 was cloned into the pMAL C2X vector. Escherichia coli cells (strain BL21) harboring the pMAL C2X-AtHSP90.1 fusion construct were grown in Luria-Bertani medium at 37°C until OD600 0.6. Subsequently, isopropyl β-D-1-thiogalactopyranoside (0.3 mM) was added to the medium and the cells were further grown for 4 h at 30°C. After harvesting by centrifugation, E. coli cells were ruptured by sonication and the MBP-tagged fusion proteins were purified using an amylose affinity resin (New England BioLabs).

Competitive Pull-Down Assay

For competitive pull-down assay, GST-MPSR1 (1 μg) was incubated with His-Δ2GFP (500 ng) in the presence of different amounts (0, 600, 1,200, and 1,800 ng) of MBP-AtHSP90.1 in pull-down buffer (0.5 M of EDTA, 0.2 M of phenylmethylsulfonyl fluoride, PI VI [AG Scientific], and 0.5% [v/v] Triton X-100) containing Ni-NTA resin for 2 h at 4°C. After extensive washing, bound proteins were visualized by immunoblot assay.

In Vitro Self-Ubiquitination Assay

An in vitro self-ubiquitination assay was conducted as described by Cho et al. (2006). GST-MPSR1 recombinant protein was incubated for 1 h in the presence or absence of E1 (200 ng of AtUBA1), E2 (200 ng of AtUBC8), 4 mM of ATP, 2 mM of creatine P (Sigma-Aldrich), creatine phosphokinase (0.01 unit; Sigma-Aldrich), Ub (20 μg of Enzo), His-Δ2GFP (500 ng), and MBP-AtHSP90.1 (500 ng). The reaction products were resolved by SDS-PAGE on 8–12% gels and transferred to polyvinylidene difluoride membranes. The blots were probed with anti-GST antibody.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: MPSR1 (At1g26800), AtHSP90.1 (AT5G52640), AtHSP90.2 (AT5G56030), AtHSP70.1 (AT3G12580), miR414 (AT1G67195), AtCHIP (AT3G07370) AGO1 (AT1G48410), HYL1 (AT1G09700), HEN1 (AT4G20910), and AtUBC10 (AT5G53300).

Supplemental Data

The following supplemental materials are available.

Footnotes

1

This work was supported by grants from the National Research Foundation (Mid-Career Researcher Program Project no. 2017R1A2B2006750 and Basic Science Research Program Project no. 2018R1A6A1A03025607), Republic of Korea.

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