Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein.
The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class I heavy chains. We have generated an enhanced green fluorescent protein-class I heavy chain (EGFP-HC) chimeric molecule to study its dislocation and degradation in US2- and US11-expressing cells. The EGFP-HC fusion is stable in control cells, but is degraded rapidly in US2- or US11-expressing cells. Proteasome inhibitors induce in a time-dependent manner the accumulation of EGFP-HC molecules in US2- and US11-expressing cells, as assessed biochemically and by cytofluorimetry of intact cells. Pulse-chase analysis and subcellular fractionation show that EGFP-HC proteins are dislocated from the endoplasmic reticulum and can be recovered as deglycosylated fluorescent intermediates in the cytosol. These results raise the possibility that dislocation of glycoproteins from the ER may not require their full unfolding.
Fiebiger
Edda
E
Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Armenise Building, Boston, MA 02115, USA.
Story
Craig
C
Ploegh
Hidde L
HL
Tortorella
Domenico
D
D000818:Animals; D001254:Astrocytoma / Q000473:pathology; D001932:Brain Neoplasms / Q000473:pathology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D005813:Genes, Synthetic; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006183:H-2 Antigens / Q000235:genetics / Q000378:metabolism*; D015789:HLA-A2 Antigen / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry; D014162:Transfection; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D015789:HLA-A2 Antigen; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C074546:US11 protein, herpesvirus; C118670:US2 protein, Varicellovirus; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex