ER stress signaling by regulated proteolysis of ATF6.
ATF6 is an endoplasmic reticulum (ER) membrane-anchored transcription factor activated by intramembrane proteolysis in the ER stress response. Upon ER stress, ATF6 is transported from the ER to the Golgi to be processed by site-1 and site-2 proteases. The trafficking is controlled by the ER chaperone BiP/GRP78. Here, we describe the experimental methods that we have used to study of ATF6 regulation in tissue culture cells. These methods were used to investigate several key steps of ATF6 activation in the ER stress response including binding and dissociation of BiP to ATF6, translocation from the ER to the Golgi and cleavage in the Golgi. In addition, luciferase reporter assays were a sensitive way to monitor ER stress and ATF6 activation. These methods were not only useful for the study ATF6 and the ER stress response, they might also help to elucidate the roles of the ER stress response in a number of human diseases involving misfolded proteins and in the differentiation of secretory tissues which require higher ER folding capacities.
Shen
Jingshi
J
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Prywes
Ron
R
D051702:Activating Transcription Factor 6; D000595:Amino Acid Sequence; D001681:Biological Assay; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; D004268:DNA-Binding Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D014157:Transcription Factors