Clustering of Peptide-Loaded MHC Class I Molecules for Endoplasmic Reticulum Export Imaged by Fluorescence Resonance Energy Transfer

Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
The Journal of Immunology (Impact Factor: 4.92). 07/2001; 166(11):6625-32. DOI: 10.4049/jimmunol.166.11.6625
Source: PubMed


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.

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Available from: Michael Edidin, Jan 13, 2014
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    • "However , it seems that only 20 to 40% of GPI-anchored proteins are organized as clusters [33]. The distribution pattern of FRET efficiency versus acceptor concentration can be used to draw conclusions on protein clustering at the cell surface [15] [19] [36]. Fig. 3B also shows the acceptor photobleaching FRET data for a positive control, consisting of a fusion between CFP and YFP GPI-anchored. "
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    • "In a random situation, acceptor co-localization with a donor is positively correlated with acceptor levels and leads to an increase in E%. Conversely, in a clustered situation, E% is largely independent of acceptor levels, and it does not decrease to zero when acceptor trends to zero [38], [39], [42]. "
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