Membrane topology of the transporter associated with antigen processing (TAP1) within an assembled functional peptide-loading complex.
The transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticular lumen for subsequent loading onto major histocompatibility complex (MHC) class I molecules. These peptide-MHC complexes are inspected at the cell surface by cytotoxic T-lymphocytes. Assembly of the functional peptide transport and loading complex depends on intra- and intermolecular packing of transmembrane helices (TMs). Here, we have examined the membrane topology of human TAP1 within an assembled and functional transport complex by cysteine-scanning mutagenesis. The accessibility of single cysteine residues facing the cytosol or endoplasmic reticular lumen was probed by a minimally invasive approach using membrane-impermeable, thiol-specific fluorophores in semipermeabilized "living" cells. TAP1 contains ten transmembrane segments, which place the N and C termini in the cytosol. The transmembrane domain consists of a translocation core of six TMs, a building block conserved among most ATP-binding cassette transporters, and a unique additional N-terminal domain of four TMs, essential for tapasin binding and assembly of the peptide-loading complex. This study provides a first map of the structural organization of the TAP machinery within the macromolecular MHCI peptide-loading complex.
Schrodt
Susanne
S
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt/Main, Germany.
Koch
Joachim
J
Tampé
Robert
R
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation / Q000235:genetics; D017920:Antiporters / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D018411:Spodoptera
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D017920:Antiporters; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; D010455:Peptides; C073119:TAP1 protein, human; C107762:tapasin