title: Aggregate formation by ERp57-deficient MHC class I peptide-loading complexes.
abstract: The endoplasmic reticulum (ER)-resident proteins TAP, tapasin and ERp57 are the core components of the major histocompatibility complex (MHC) class I peptide-loading complex and play an important role in peptide loading by MHC class I-beta(2)microglobulin dimers. ERp57 and tapasin form a stable disulfide-linked heterodimer within the peptide-loading complex. We demonstrate that ERp57-deficient loading complexes, obtained by expression in a tapasin-negative cell line of a tapasin mutant (C95A) that is not able to form a disulfide bond with ERp57, are prone to aggregation. We studied the assembly, stability and aggregation of the core loading complex using cell lines stably expressing fluorescently tagged tapasin (wild type or C95A mutant) and TAP1. Part of the loading complexes containing the tagged C95A tapasin and TAP1 were sequestered in the ER, without change of their ER transmembrane topology, and were surrounded by a mesh of filaments at the cytosolic side, resulting in formation of protein aggregates with characteristic morphology. Protein aggregates were associated with changes in ER protein turnover but did not affect the cell viability and did not induce the unfolded protein response. Fluorescence resonance energy transfer analysis of the aggregate-free ER fraction revealed that lack of ERp57 did not affect the stoichiometry or stability of tapasin-TAP1 interactions in the assembled 'soluble' core loading complexes. We conclude that the presence of ERp57 is important for the stability of core loading complexes, and that in its absence, the core loading complexes may form stable aggregates within the ER.
authors.affiliation: Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520, USA.
authors.forename: David
authors.initials: D
authors.lastname: Stepensky
authors.forename: Naveen
authors.initials: N
authors.lastname: Bangia
authors.forename: Peter
authors.initials: P
authors.lastname: Cresswell
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D004220:Disulfides / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000737:chemistry*; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction