Abstract
Tapasin organizes the peptide-loading complex (PLC) by recruiting peptide-receptive MHC class I (MHC-I) and accessory chaperones to the N-terminal regions of the TAP subunits TAP1 and TAP2. Despite numerous studies have shown that the formation of the PLC is essential to facilitate proper MHC-I loading, the molecular architecture of this complex is still highly controversial. We studied the stoichiometry of the PLC by blue native-PAGE in combination with Ab-shift assays and found that TAP/tapasin complexes exist at steady state as a mixture of two distinct oligomers of 350 and 450 kDa. Only the higher m.w. complex contains MHC-I and disulfide-linked tapasin/ER60 conjugates. Moreover, we show for the first time to our knowledge that the fully assembled PLC comprises two tapasin, two ER60, but only one complex of MHC-I and calreticulin. Based hereon we postulate that the TAP subunits alternate in the recruitment and loading of a single MHC-I.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP Binding Cassette Transporter, Subfamily B, Member 2
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ATP Binding Cassette Transporter, Subfamily B, Member 3
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ATP-Binding Cassette Transporters / genetics
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ATP-Binding Cassette Transporters / metabolism*
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Alternative Splicing / genetics
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Binding Sites
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Calreticulin / metabolism
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Cell Line
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Dimerization
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Disulfides / metabolism
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Histocompatibility Antigens Class I / metabolism*
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Humans
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Membrane Transport Proteins / metabolism
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Molecular Chaperones / metabolism
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Peptides / metabolism*
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Protein Binding
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Protein Disulfide-Isomerases / metabolism
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Type C Phospholipases / metabolism
Substances
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ATP Binding Cassette Transporter, Subfamily B, Member 2
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ATP Binding Cassette Transporter, Subfamily B, Member 3
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ATP-Binding Cassette Transporters
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Calreticulin
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Disulfides
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Histocompatibility Antigens Class I
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Membrane Transport Proteins
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Molecular Chaperones
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Peptides
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TAP1 protein, human
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Tap1 protein, rat
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Tap2 protein, rat
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tapasin
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TAP2 protein, human
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Type C Phospholipases
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Protein Disulfide-Isomerases