FUNCTIONAL CHARACTERIZATION OF PROTEIN DISULFIDE ISOMERASE 9 IN ARABIDOPSIS THALIANA AND ITS ROLE IN THE UNFOLDED PROTEIN RESPONSE
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2024
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Abstract
The endoplasmic reticulum (ER) is responsible for synthesis, folding, and maturation of most secretory proteins in eukaryotes. Environmental stressors disrupt secretory protein folding and proteostasis in the ER leading to ER stress. The unfolded protein response (UPR) senses ER stress and restores proteostasis by increasing the expression of ER resident protein folding chaperones, such as the protein disulfide isomerases (PDIs) and BiP. In plants, the transmembrane ER stress sensor-kinase, IRE1, activates the UPR by unconventionally splicing the mRNA encoding the bZIP60 transcription factor, triggering UPR gene transcription. The induced PDIs catalyze disulfide-based polypeptide folding to restore the folding capacity in the ER. The Arabidopsis PDI-M subfamily member, PDI9, is highly induced in response to ER stress and the UPR, however the substrates with which PDI9 interact and the specific role that PDI9 has on the ER stress response is not well understood. To determine the modulatory role of PDI9 on the UPR, a bZIP60 reporter construct fused to GFP was generated to measure UPR activation. Interaction with the conserved ER stress sensor IRE1 was also tested. Through various protoplast transfection assays and quantitative microscopy analyses, it was revealed that PDI9 modulated the UPR and interacts with IRE1 in Arabidopsis. Genetic markers of UPR were highly upregulated in the pdi9 mutants relative to WT, suggesting PDI9 plays an important role in mitigating against overactive UPR and in maintaining proteostasis under stress. To identify other novel protein interactors of PDI9, a PDI9 stable transformant was generated and pull-down experiments were performed from ER-stress induced roots and cotyledons of Arabidopsis. It was discovered that PDI9 interacts with the UPR-mediator and ER-resident molecular chaperones, BiP1 and BiP2. The dynamin-related GTPases, DRP1 and DRP2, were also discovered as novel interactors of PDI9. In addition, homodimerization was detected between PDI9 through quantitative FRET analysis in protoplast cells. These findings indicate that PDI9 plays an important role in the UPR pathway through interaction with IRE1 to potentially attenuate the UPR in a manner analogous to mammalian models. Additional regulatory roles of PDI9 may also be taking place to modulate this UPR pathway through interaction with the UPR-responsive chaperone, BiP1 and BiP2, or by PDI9 homodimerization. We propose a model in which PDI9 modulates the UPR through two competing activities: secretory protein folding and via interaction with IRE1 to maintain proteostasis in plants. By uncovering novel biological roles of PDIs and regulatory players of the UPR in plants, these results contribute to the foundational understanding of how plants mitigate and adapt to environmental stressors such as heat and drought, to maintain their viability and growth.
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Plant sciences, Agriculture, Molecular biology, ER stress, Heat stress, Protein disulfide isomerase (PDI), Protein folding, Unfolded protein response (UPR)
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167 pages
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