In vitro reconstitution of ER-stress induced ATF6 transport in COPII vesicles.
The transcription factor ATF6 is held as a membrane precursor in the endoplasmic reticulum (ER), and is transported and proteolytically processed in the Golgi apparatus under conditions of unfolded protein response stress. We show that during stress, ATF6 forms an interaction with COPII, the protein complex required for vesicular traffic of cargo proteins from the ER. Using an in vitro budding reaction that recapitulates the ER-stress induced transport of ATF6, we show that no cytoplasmic proteins other than COPII are necessary for transport. ATF6 is retained in the ER by association with the chaperone BiP (GRP78). In the in vitro reaction, the ATF6-BiP complex disassembles when membranes are treated with reducing agent and ATP. A hybrid protein with the ATF6 cytoplasmic domain replaced by a constitutive sorting signal (Sec22b SNARE) retains stress-responsive transport in vivo and in vitro. These results suggest that unfolded proteins or an ER luminal -SH reactive bond controls BiP-ATF6 stability and access of ATF6 to the COPII budding machinery.
Schindler
Adam J
AJ
Department of Molecular and Cell Biology and Howard Hughes Medical Institute, Barker Hall, University of California, Berkeley, CA 94720-3202, USA.
Schekman
Randy
R
D051702:Activating Transcription Factor 6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D022181:COP-Coated Vesicles / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects
C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C484373:SEC22A protein, human; D000255:Adenosine Triphosphate; D004229:Dithiothreitol