ABSTRACT
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.
Subject(s)
Acetylcysteine/analogs & derivatives , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , HLA-A2 Antigen/metabolism , Peptide Fragments/immunology , Peptide Fragments/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/metabolism , Acetylcysteine/pharmacology , Amino Acid Sequence , Antigen Presentation/drug effects , Antigen Presentation/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Cell Line, Transformed , Cysteine Proteinase Inhibitors/pharmacology , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/genetics , Energy Transfer/drug effects , Energy Transfer/genetics , Energy Transfer/immunology , Gene Products, tax/immunology , Gene Products, tax/metabolism , Green Fluorescent Proteins , HLA-A2 Antigen/genetics , HeLa Cells , Human T-lymphotropic virus 1/immunology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/immunology , Luminescent Proteins/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/genetics , Protein Transport/drug effects , Protein Transport/genetics , Protein Transport/immunology , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Spectrometry, FluorescenceABSTRACT
Lateral diffusion of GFP-tagged H2Ld molecules in the ER membrane reports on their interaction with the TAP complex during synthesis and peptide loading. Peptide-loaded H2Ld molecules diffuse rapidly, near the theoretical limit for proteins in a bilayer. However, these molecules are retained in the ER for some time after assembly. H2Ld molecules, associated with the TAP complex, diffuse slowly, as does GFP-tagged TAP1. This implies that the association of H2Ld molecules with the TAP complex is stable for at least several minutes. It also suggests that the TAP complex is very large, perhaps containing hundreds of proteins.
