Clustering of peptide-loaded MHC class I molecules for endoplasmic reticulum export imaged by fluorescence resonance energy transfer.
Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.
Pentcheva
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Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Edidin
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D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D017951:Antigen Presentation / Q000187:drug effects / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D004735:Energy Transfer / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D016356:Gene Products, tax / Q000276:immunology / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015789:HLA-A2 Antigen / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D015368:Human T-lymphotropic virus 1 / Q000276:immunology; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D015853:Cysteine Proteinase Inhibitors; D016356:Gene Products, tax; D015789:HLA-A2 Antigen; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C073119:TAP1 protein, human; C409504:blue fluorescent protein, Aequorea victoria; C054562:yellow fluorescent protein, Bacteria; C067713:lactacystin; D049452:Green Fluorescent Proteins; D000111:Acetylcysteine