title: The murine cytomegalovirus pp89 immunodominant H-2Ld epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide.
abstract: The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L(d) 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.
authors.affiliation: Institute of Biochemistry, Medical Faculty, Charité, Humboldt University, Monbijoustrasse 2a, 10117 Berlin, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Knuehl
authors.forename: P
authors.initials: P
authors.lastname: Spee
authors.forename: T
authors.initials: T
authors.lastname: Ruppert
authors.forename: U
authors.initials: U
authors.lastname: Kuckelkorn
authors.forename: P
authors.initials: P
authors.lastname: Henklein
authors.forename: J
authors.initials: J
authors.lastname: Neefjes
authors.forename: P M
authors.initials: PM
authors.lastname: Kloetzel
mesh_terms: 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; D017951:Antigen Presentation; D001324:Autoantigens; D001693:Biological Transport, Active / Q000276:immunology; D018797:Cell Cycle Proteins; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D006183:H-2 Antigens / Q000096:biosynthesis* / Q000378:metabolism; D064532:Histocompatibility Antigen H-2D; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis* / Q000378:metabolism; D016056:Immunodominant Epitopes / Q000096:biosynthesis* / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism; D018146:Muromegalovirus / Q000276:immunology*; D009124:Muscle Proteins; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011498:Protein Precursors / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology; D051381:Rats
chemical_list: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D001324:Autoantigens; D018797:Cell Cycle Proteins; D006183:H-2 Antigens; D064532:Histocompatibility Antigen H-2D; D017874:Immediate-Early Proteins; D016056:Immunodominant Epitopes; C099558:Ki antigen; D009097:Multienzyme Complexes; D009124:Muscle Proteins; C085047:PSME1 protein, human; D010455:Peptides; D011498:Protein Precursors; D011506:Proteins; C490050:Psme1 protein, rat; C490052:Psme2 protein, rat; C073119:TAP1 protein, human; C486171:Tap1 protein, mouse; C486172:Tap1 protein, rat; C489516:Tap2 protein, mouse; C489514:Tap2 protein, rat; C052612:cytomegalovirus immediate early phosphoprotein pp89; C071692:TAP2 protein, human; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex