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MBoC | ARTICLE Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

By Patrick Lajoiea, Robyn D. Moirb and Ian M. Willisb


ABSTRACT Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, includ-ing BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous re-porter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recov-ery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secre-tory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of un-folded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. Dur-ing adaptation, we observe a significant lag between down-regulation of the UPR and resolu-tion of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of dis-tinguishing between the different mechanisms leading to UPR activation in living cells

Year: 2012
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