Live cell imaging of protein dislocation from the endoplasmic reticulum.
Misfolded proteins in the endoplasmic reticulum (ER) are dislocated to the cytosol to be degraded by the proteasomes. Various plant and bacterial toxins and certain viruses hijack this dislocation pathway to exert their toxicity or to infect cells. In this study, we report a dislocation-dependent reconstituted GFP (drGFP) assay that allows, for the first time, imaging proteins dislocated from the ER lumen to the cytosol in living cells. Our results indicate that both luminal and membrane-spanning ER proteins can be fully dislocated from the ER to the cytosol. By combining the drGFP assay with RNAi or chemical inhibitors of proteins in the Hrd1 ubiquitin ligase complex, we demonstrate that the Sel1L, Hrd1, p97/VCP, and importin β proteins are required for the dislocation of misfolded luminal α-1 antitrypsin. The strategy described in this work is broadly applicable to the study of other types of transmembrane transport of proteins and likely also of viruses and toxins in living cells.
Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Yongwang
Y
Zhong
Shengyun
S
Fang
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000502:physiology; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D000074405:Valosin Containing Protein; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D011506:Proteins; C117384:SEL1L protein, human; D000515:alpha 1-Antitrypsin; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins; C482345:SYVN1 protein, human; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein