(100-27) Protein Disulfide Isomerase-9 modulates cellular stress in Arabidopsis through two mechanisms: Protein folding and via interaction with the master regulator, IRE1
Graduate student, Molecular Biosciences and Bioengineering University of Hawaii Honolulu, Hawaii
Body of Abstract: Protein disulfide isomerases (PDIs) facilitate protein folding in the endoplasmic reticulum (ER) by catalyzing the formation and rearrangement of disulfide bonds in protein substrates. When environmental stresses lead to an accumulation of unfolded proteins, ER stress occurs. PDIs facilitate the mitigation of ER stress via the unfolded protein response (UPR). We previously showed that the expression of a unique member of the PDI-family, PDI9, is stimulated by the UPR-induced transcription factor, bZIP60. ER-localized PDI9 was also shown to be essential to form disulfide-bonds during protein folding and for pollen cell wall biogenesis during heat stress.We further investigated the mechanisms by which PDI9 participates in the UPR. We showed that PDI9 interacts with the ER membrane sensor and master regulator of the UPR pathway, inositol-requiring enzyme 1 (IRE1). PDI9-IRE1 interactions were measured by coimmunoprecipitation of recombinant tagged PDI9 and IRE1 in vitro and by using laser scanning confocal microscopy to measure Fluorescence Lifetime and Fluorescence Resonance Energy Transfer (FLIM-FRET) between IRE1-RFP and PDI9-GFP fusions expressed in leaf mesophyll protoplasts in vivo. Site-specific mutagenesis was used to determine interaction point residues of PDI9 and IRE1. The PDI9-RFP fusion co-localizes with IRE1-GFP fusion in the ER and interacts at key cysteine residues with statistically significant FRET efficiencies of 5.9 - 6.8%. PDI9 knockout mutants exhibit a hyperstimulation of UPR as measured by qPCR of UPR markers and by transient expression of the downstream UPR biosensors (the PDI9-promoter-RFP and the bZIP60-intron-GFP constructs), in mesophyll protoplasts. Our results support the hypothesis that the pool of PDI9 acts to modulate the UPR pathway via two mechanisms: a) the loss of PDI9-based protein folding stimulates UPR; and b) PDI9 directly interacts with IRE1 to modulate sensor output.