title: 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.
abstract: Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes under both physiological and stress conditions. In Arabidopsis, there are seven HSP90 isoforms (HSP90.1-HSP90.7) that are localized in the cytoplasm/nucleus, mitochondrion, chloroplast, and endoplasmic reticulum (ER) where protein folding actively takes place. In this study, we analysed the sequence of ER-localized Arabidopsis HSP90.7 and the other ER GRP94 proteins from plants and animals, and identified a short, charged region that is specifically present in the middle domain of plant-derived GRP94 proteins. To understand the role of this charged region, we analysed transgenic plants that expressed a mutant protein, HSP90.7(Δ22), which had this charged region deleted. 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.
authors.affiliation: Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada M1C 1A4.
authors.forename: Lisa P
authors.initials: LP
authors.lastname: Chong
authors.affiliation: Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada M1C 1A4.
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada M1C 1A4.
authors.forename: Nanette
authors.initials: N
authors.lastname: Gad
authors.affiliation: Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada M1C 1A4.
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Anderson
authors.affiliation: Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada M1C 1A4.
authors.forename: Bhavank
authors.initials: B
authors.lastname: Shah
authors.affiliation: Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada M1C 1A4 rzhao@utsc.utoronto.ca.
authors.forename: Rongmin
authors.initials: R
authors.lastname: Zhao
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017360:Arabidopsis / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D016415:Sequence Alignment; D013312:Stress, Physiological / Q000235:genetics*; D014415:Tunicamycin / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D029681:Arabidopsis Proteins; D018841:HSP90 Heat-Shock Proteins; C543881:Hsp90-7 protein, Arabidopsis; D014415:Tunicamycin; D002118:Calcium
keywords: ER stress responses; Enzyme kinetics; HSP90 client proteins; molecular chaperone; protein–protein interaction; transgenic plant.