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A highly charged region in the middle domain of plant endoplasmic reticulum (ER)-localized heat-shock protein 90 is required for resistance to tunicamycin or high calcium-induced ER stresses.

Chong LP, Wang Y, Gad N, Anderson N, Shah B, Zhao R - J. Exp. Bot. (2014)

Bottom Line: We showed that seedlings expressing HSP90.7(Δ22) had significantly enhanced sensitivity to ER stress induced by tunicamycin or a high concentration of calcium, although its general chaperone activity in preventing the model protein from heat-induced aggregation was not significantly affected.We also analysed the ATP-binding and hydrolysis activity of both wild-type and mutant HSP90.7 proteins, and found that they had slightly different ATP-binding affinities.Finally, using a yeast two-hybrid screen, we identified a small set of HSP90.7 interactors and showed that the charged region is not required for the candidate client interaction, although it may affect their binding affinity, thus providing potential targets for further investigation of HSP90.7 functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada M1C 1A4.

No MeSH data available.


Immunoblotting of representative ER-localized chaperones and FLAG-tagged HSP90.7 complexes. (A) Top panel: total cell lysate of 2-week-old transgenic seedlings expressing HSP90.7Δ22 was immunoblotted with anti-FLAG, anti-HSP90.7, anti-HSP90.2, anti-calnexin, and anti-BiP antibodies. Bottom panel: 8-d-old seedlings grown on ½ strength MS agar plates were treated with 5 μM tunicamycin or 2mM DTT in liquid ½ strength MS salt for 8h, and the total proteins were then immunoblotted with anti-BiP antibody. Treatments with DMSO or H2O only were used as negative controls. (B) FLAG-tagged HSP90.7 or HSP90.7Δ22 complexes were purified using anti-FLAG antibody resin and resolved by 12% SDS-PAGE followed by silver staining (top). The bottom panel shows immunoblotting of the same samples with anti-HSP90.7 antibody. The arrows indicate the FLAG-tagged HSP90.7 or degradation intermediates. (C) Yeast EGY48 cells carrying prey and bait plasmids, which were originally diluted to an OD600 of 1.0 and 0.1 and applied to synthetic medium supplemented with galactose but without uracil, histidine, tryptophan, and leucine (SGal–UHWL), were grown for 7 d at 30 °C. pEG202-RFMH1 acted as a negative control. (D) Immunoblotting analysis of prey protein expression in yeast EGY48 cells carrying bait plasmid pEG202-HSP90.7MC and prey plasmids for At4G21960.1, At4G24730.2, and At1G20330.1, as indicated. Anti-HA antibody for prey protein and anti-HSP90.7 antibody for bait protein were used.
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Figure 7: Immunoblotting of representative ER-localized chaperones and FLAG-tagged HSP90.7 complexes. (A) Top panel: total cell lysate of 2-week-old transgenic seedlings expressing HSP90.7Δ22 was immunoblotted with anti-FLAG, anti-HSP90.7, anti-HSP90.2, anti-calnexin, and anti-BiP antibodies. Bottom panel: 8-d-old seedlings grown on ½ strength MS agar plates were treated with 5 μM tunicamycin or 2mM DTT in liquid ½ strength MS salt for 8h, and the total proteins were then immunoblotted with anti-BiP antibody. Treatments with DMSO or H2O only were used as negative controls. (B) FLAG-tagged HSP90.7 or HSP90.7Δ22 complexes were purified using anti-FLAG antibody resin and resolved by 12% SDS-PAGE followed by silver staining (top). The bottom panel shows immunoblotting of the same samples with anti-HSP90.7 antibody. The arrows indicate the FLAG-tagged HSP90.7 or degradation intermediates. (C) Yeast EGY48 cells carrying prey and bait plasmids, which were originally diluted to an OD600 of 1.0 and 0.1 and applied to synthetic medium supplemented with galactose but without uracil, histidine, tryptophan, and leucine (SGal–UHWL), were grown for 7 d at 30 °C. pEG202-RFMH1 acted as a negative control. (D) Immunoblotting analysis of prey protein expression in yeast EGY48 cells carrying bait plasmid pEG202-HSP90.7MC and prey plasmids for At4G21960.1, At4G24730.2, and At1G20330.1, as indicated. Anti-HA antibody for prey protein and anti-HSP90.7 antibody for bait protein were used.

Mentions: To further understand whether the enhanced sensitivity to tunicamycin and a high concentration of Ca2+ for HSP90.7Δ22 expression seedlings was due to excessive ER stress, we analysed the protein level of BiP, whose overexpression is a hallmark for ER stress (Gardner et al., 2013). Interestingly, the expression level of BiP was not upregulated in FLAG-tagged HSP90.7Δ22 transgenic seedlings compared with wild type (Fig. 7A, top panel). The expression of cytosolic HSP90 or ER-chaperone calnexin was also not affected in transgenic HSP90.7Δ22 seedlings (Fig. 7A). This suggested that the general protein homeostasis in both the cytosol and the ER lumen might not be significantly affected under normal growth conditions. We also tested whether expression of HSP90.7Δ22 affected the UPR if treated with tunicamycin or DTT, the two commonly used UPR-inducing reagents. We noticed that both tunicamycin and DTT induced the expression of BiP well; however, there is no difference between wild type and HSP90.7Δ22 expression lines (Fig. 7A bottom panel), suggesting that expression of HSP90.7Δ22 might not significantly affect the normal UPR, at least not for the induction of BiP under ER stress. In an attempt to understand whether deletion of the charged region interfered with the association of any specific HSP90.7 clients, we purified both FLAG-tagged HSP90.7 and HSP90.7Δ22 complexes by affinity purification. Although both FLAG-tagged HSP90.7 and HSP90.7Δ22 were purified well, no other protein was significantly co-purified as shown by SDS-PAGE and silver staining (Fig. 7B). We tried to analyse the HSP90.7FLAG and HSP90.7Δ22FLAG protein complexes by liquid chromatography/tandem mass spectrometry; however, no robust HSP90.7 binding partners were identified (data not shown). This is presumably because client proteins bind HSP90.7 so weakly that the HSP90.7 protein complex was not well preserved in the affinity purification.


A highly charged region in the middle domain of plant endoplasmic reticulum (ER)-localized heat-shock protein 90 is required for resistance to tunicamycin or high calcium-induced ER stresses.

Chong LP, Wang Y, Gad N, Anderson N, Shah B, Zhao R - J. Exp. Bot. (2014)

Immunoblotting of representative ER-localized chaperones and FLAG-tagged HSP90.7 complexes. (A) Top panel: total cell lysate of 2-week-old transgenic seedlings expressing HSP90.7Δ22 was immunoblotted with anti-FLAG, anti-HSP90.7, anti-HSP90.2, anti-calnexin, and anti-BiP antibodies. Bottom panel: 8-d-old seedlings grown on ½ strength MS agar plates were treated with 5 μM tunicamycin or 2mM DTT in liquid ½ strength MS salt for 8h, and the total proteins were then immunoblotted with anti-BiP antibody. Treatments with DMSO or H2O only were used as negative controls. (B) FLAG-tagged HSP90.7 or HSP90.7Δ22 complexes were purified using anti-FLAG antibody resin and resolved by 12% SDS-PAGE followed by silver staining (top). The bottom panel shows immunoblotting of the same samples with anti-HSP90.7 antibody. The arrows indicate the FLAG-tagged HSP90.7 or degradation intermediates. (C) Yeast EGY48 cells carrying prey and bait plasmids, which were originally diluted to an OD600 of 1.0 and 0.1 and applied to synthetic medium supplemented with galactose but without uracil, histidine, tryptophan, and leucine (SGal–UHWL), were grown for 7 d at 30 °C. pEG202-RFMH1 acted as a negative control. (D) Immunoblotting analysis of prey protein expression in yeast EGY48 cells carrying bait plasmid pEG202-HSP90.7MC and prey plasmids for At4G21960.1, At4G24730.2, and At1G20330.1, as indicated. Anti-HA antibody for prey protein and anti-HSP90.7 antibody for bait protein were used.
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Figure 7: Immunoblotting of representative ER-localized chaperones and FLAG-tagged HSP90.7 complexes. (A) Top panel: total cell lysate of 2-week-old transgenic seedlings expressing HSP90.7Δ22 was immunoblotted with anti-FLAG, anti-HSP90.7, anti-HSP90.2, anti-calnexin, and anti-BiP antibodies. Bottom panel: 8-d-old seedlings grown on ½ strength MS agar plates were treated with 5 μM tunicamycin or 2mM DTT in liquid ½ strength MS salt for 8h, and the total proteins were then immunoblotted with anti-BiP antibody. Treatments with DMSO or H2O only were used as negative controls. (B) FLAG-tagged HSP90.7 or HSP90.7Δ22 complexes were purified using anti-FLAG antibody resin and resolved by 12% SDS-PAGE followed by silver staining (top). The bottom panel shows immunoblotting of the same samples with anti-HSP90.7 antibody. The arrows indicate the FLAG-tagged HSP90.7 or degradation intermediates. (C) Yeast EGY48 cells carrying prey and bait plasmids, which were originally diluted to an OD600 of 1.0 and 0.1 and applied to synthetic medium supplemented with galactose but without uracil, histidine, tryptophan, and leucine (SGal–UHWL), were grown for 7 d at 30 °C. pEG202-RFMH1 acted as a negative control. (D) Immunoblotting analysis of prey protein expression in yeast EGY48 cells carrying bait plasmid pEG202-HSP90.7MC and prey plasmids for At4G21960.1, At4G24730.2, and At1G20330.1, as indicated. Anti-HA antibody for prey protein and anti-HSP90.7 antibody for bait protein were used.
Mentions: To further understand whether the enhanced sensitivity to tunicamycin and a high concentration of Ca2+ for HSP90.7Δ22 expression seedlings was due to excessive ER stress, we analysed the protein level of BiP, whose overexpression is a hallmark for ER stress (Gardner et al., 2013). Interestingly, the expression level of BiP was not upregulated in FLAG-tagged HSP90.7Δ22 transgenic seedlings compared with wild type (Fig. 7A, top panel). The expression of cytosolic HSP90 or ER-chaperone calnexin was also not affected in transgenic HSP90.7Δ22 seedlings (Fig. 7A). This suggested that the general protein homeostasis in both the cytosol and the ER lumen might not be significantly affected under normal growth conditions. We also tested whether expression of HSP90.7Δ22 affected the UPR if treated with tunicamycin or DTT, the two commonly used UPR-inducing reagents. We noticed that both tunicamycin and DTT induced the expression of BiP well; however, there is no difference between wild type and HSP90.7Δ22 expression lines (Fig. 7A bottom panel), suggesting that expression of HSP90.7Δ22 might not significantly affect the normal UPR, at least not for the induction of BiP under ER stress. In an attempt to understand whether deletion of the charged region interfered with the association of any specific HSP90.7 clients, we purified both FLAG-tagged HSP90.7 and HSP90.7Δ22 complexes by affinity purification. Although both FLAG-tagged HSP90.7 and HSP90.7Δ22 were purified well, no other protein was significantly co-purified as shown by SDS-PAGE and silver staining (Fig. 7B). We tried to analyse the HSP90.7FLAG and HSP90.7Δ22FLAG protein complexes by liquid chromatography/tandem mass spectrometry; however, no robust HSP90.7 binding partners were identified (data not shown). This is presumably because client proteins bind HSP90.7 so weakly that the HSP90.7 protein complex was not well preserved in the affinity purification.

Bottom Line: We showed that seedlings expressing HSP90.7(Δ22) had significantly enhanced sensitivity to ER stress induced by tunicamycin or a high concentration of calcium, although its general chaperone activity in preventing the model protein from heat-induced aggregation was not significantly affected.We also analysed the ATP-binding and hydrolysis activity of both wild-type and mutant HSP90.7 proteins, and found that they had slightly different ATP-binding affinities.Finally, using a yeast two-hybrid screen, we identified a small set of HSP90.7 interactors and showed that the charged region is not required for the candidate client interaction, although it may affect their binding affinity, thus providing potential targets for further investigation of HSP90.7 functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada M1C 1A4.

No MeSH data available.