biostudies-literature00470Rizvi SMNIAID NIH HHS18220-5https://www.ebi.ac.uk/biostudies/studies/S-EPMC1838733biostudies-literatureUnknown103(48)Tapasin (Tpn) has been implicated in multiple steps of the MHC class I assembly pathway, but the mechanisms of function remain incompletely understood. Using purified proteins, we could demonstrate direct binding of Tpn to peptide-deficient forms of MHC class I molecules at physiological temperatures. Tpn also bound to M10.5, a pheromone receptor-associated MHC molecule that has an open and empty groove and that shares significant sequence identity with class I sequences. Two types of MHC class I-Tpn complexes were detectable in vitro depending on the input proteins; those depleted in beta(2)m, and those containing beta(2)m. Both were competent for subsequent assembly with peptides, but the latter complexes assembled more rapidly. Thus, the assembly rate of Tpn-associated class I was determined by the conditions under which Tpn-MHC class I complexes were induced. Peptide loading of class I inhibited Tpn-class I-binding interactions, and peptide-depletion enhanced binding. In combination with beta(2)m, certain peptides induced efficient dissociation of preformed Tpn-class I complexes. Together, these studies demonstrate direct Tpn-MHC class I interactions and preferential binding of empty MHC class I by Tpn, and that the Tpn-class I interaction is regulated by both beta(2)m and peptide. In cells, Tpn is likely to be a direct mediator of peptide-regulated binding and release of MHC class I from the TAP complex.Proceedings of the National Academy of Sciences of the United States of AmericaDirect peptide-regulatable interactions between MHC class I molecules and tapasin.PMC1838733AI-44115R56 AI044115R01 AI044115Raghavan MRizvi SM47falseDirect peptide-regulatable interactions between MHC class I molecules and tapasin.Tapasin (Tpn) has been implicated in multiple steps of the MHC class I assembly pathway, but the mechanisms of function remain incompletely understood. Using purified proteins, we could demonstrate direct binding of Tpn to peptide-deficient forms of MHC class I molecules at physiological temperatures. Tpn also bound to M10.5, a pheromone receptor-associated MHC molecule that has an open and empty groove and that shares significant sequence identity with class I sequences. Two types of MHC class I-Tpn complexes were detectable in vitro depending on the input proteins; those depleted in beta(2)m, and those containing beta(2)m. Both were competent for subsequent assembly with peptides, but the latter complexes assembled more rapidly. Thus, the assembly rate of Tpn-associated class I was determined by the conditions under which Tpn-MHC class I complexes were induced. Peptide loading of class I inhibited Tpn-class I-binding interactions, and peptide-depletion enhanced binding. In combination with beta(2)m, certain peptides induced efficient dissociation of preformed Tpn-class I complexes. Together, these studies demonstrate direct Tpn-MHC class I interactions and preferential binding of empty MHC class I by Tpn, and that the Tpn-class I interaction is regulated by both beta(2)m and peptide. In cells, Tpn is likely to be a direct mediator of peptide-regulated binding and release of MHC class I from the TAP complex.2006-01-01T00:00:00Z2006 Nov2021-03-12T08:25:26Z2019-03-27T02:03:39ZS-EPMC18387331711688410.1073/pnas.0605131103