Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells
Open Access
- 1 March 2012
- journal article
- Published by American Society for Cell Biology (ASCB) in Molecular Biology of the Cell
- Vol. 23 (5), 955-964
- https://doi.org/10.1091/mbc.e11-12-0995
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, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter 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 recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution 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 distinguishing between the different mechanisms leading to UPR activation in living cells.Keywords
This publication has 57 references indexed in Scilit:
- Protein folding and quality control in the endoplasmic reticulum: Recent lessons from yeast and mammalian cell systemsCurrent Opinion in Cell Biology, 2011
- Disulfide bonds in ER protein folding and homeostasisCurrent Opinion in Cell Biology, 2011
- Fluorescent proteins: a cell biologist's user guideTrends in Cell Biology, 2009
- Messenger RNA targeting to endoplasmic reticulum stress signalling sitesNature, 2008
- Real-Time Redox Measurements during Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding FunctionsCell, 2008
- Engineering and characterization of a superfolder green fluorescent proteinNature Biotechnology, 2005
- A versatile toolbox for PCR‐based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettesYeast, 2004
- Global analysis of protein localization in budding yeastNature, 2003
- Block of HAC1 mRNA Translation by Long-Range Base Pairing Is Released by Cytoplasmic Splicing upon Induction of the Unfolded Protein ResponseCell, 2001
- A Novel Mechanism for Regulating Activity of a Transcription Factor That Controls the Unfolded Protein ResponseCell, 1996