Functional dissection of the transmembrane domains of the transporter associated with antigen processing (TAP).
The transporter associated with antigen processing (TAP1/2) translocates cytosolic peptides of proteasomal degradation into the endoplasmic reticulum (ER) lumen. A peptide-loading complex of tapasin, major histocompatibility complex class I, and several auxiliary factors is assembled at the transporter to optimize antigen display to cytotoxic T-lymphocytes at the cell surface. The heterodimeric TAP complex has unique N-terminal domains in addition to a 6 + 6-transmembrane segment core common to most ABC transporters. Here we provide direct evidence that this core TAP complex is sufficient for (i) ER targeting, (ii) heterodimeric assembly within the ER membrane, (iii) peptide binding, (iv) peptide transport, and (v) specific inhibition by the herpes simplex virus protein ICP47 and the human cytomegalovirus protein US6. We show for the first time that the translocation pore of the transporter is composed of the predicted TM-(5-10) of TAP1 and TM-(4-9) of TAP2. Moreover, we demonstrate that the N-terminal domains of TAP1 and TAP2 are essential for recruitment of tapasin, consequently mediating assembly of the macromolecular peptide-loading complex.
Koch
Joachim
J
Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University Frankfurt, Germany.
Guntrum
Renate
R
Heintke
Susanne
S
Kyritsis
Christoph
C
Tampé
Robert
R
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D017920:Antiporters / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005805:Genes, MHC Class I; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000502:physiology; D015151:Immunoblotting; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism; D007313:Insecta; D026901:Membrane Transport Proteins; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012685:Sepharose / Q000737:chemistry
D018528:ATP-Binding Cassette Transporters; D017920:Antiporters; D015395:Histocompatibility Antigens Class I; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes; D010455:Peptides; C107762:tapasin; C477327:transporter associated with antigen processing (TAP); D000255:Adenosine Triphosphate; D012685:Sepharose; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex