Novel peptide-binding proteins and peptide transport in normal and TAP-deficient microsomes.
Most major histocompatibility complex (MHC) class I-binding peptides are translocated by TAP heterodimers, but some enter the ER lumen by alternative pathways. To further define mechanisms of peptide handling, we developed a system for the analysis of peptide-binding components in the ER membrane and lumen using iodinated cross-linkable peptide derivatives. Here we demonstrate that at least three proteins bind peptides in the ER lumen. Peptide cross-linking to these lumenal proteins can be used as an alternative method to monitor peptide transport. TAP and one other protein bind peptides on the cytoplasmic face of the ER. The presence of multiple peptide-binding proteins necessitates caution in interpreting traditional peptide-binding and transport assays. Finally, we demonstrate sequence-specific peptide transport in TAP-deficient cells transfected with only rat TAP1.
Marusina
K
K
Howard Hughes Medical Institute, Department of Molecular Genetics, University of Cincinnati, Ohio 45267-0524, USA.
Reid
G
G
Gabathuler
R
R
Jefferies
W
W
Monaco
J J
JJ
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D002460:Cell Line; D003432:Cross-Linking Reagents; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000276:immunology / Q000378:metabolism*; D008862:Microsomes, Liver / Q000276:immunology / Q000378:metabolism; D008970:Molecular Weight; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D003432:Cross-Linking Reagents; D015395:Histocompatibility Antigens Class I; D009842:Oligopeptides; D011506:Proteins; C073119:TAP1 protein, human; C486171:Tap1 protein, mouse; C486172:Tap1 protein, rat; C489516:Tap2 protein, mouse; C489514:Tap2 protein, rat; C071692:TAP2 protein, human