Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter.
The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Street 9, D-60438 Frankfurt/M., Germany.
Nina
N
Grossmann
Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, USA.
Ahmet S
AS
Vakkasoglu
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Street 9, D-60438 Frankfurt/M., Germany.
Sabine
S
Hulpke
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Street 9, D-60438 Frankfurt/M., Germany.
Rupert
R
Abele
Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, USA.
Rachelle
R
Gaudet
1] Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Street 9, D-60438 Frankfurt/M., Germany [2] Cluster of Excellence Frankfurt-Macromolecular Complexes, Goethe-University Frankfurt, Max-von-Laue-Street 9, D-60438 Frankfurt/M., Germany.
Robert
R
Tampé
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017951:Antigen Presentation / Q000502:physiology*; D000941:Antigens / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D019281:Dimerization; D006868:Hydrolysis; D007313:Insecta; D023421:Models, Animal; D000069284:Protein Translocation Systems / Q000378:metabolism*; D051381:Rats
D018528:ATP-Binding Cassette Transporters; D000941:Antigens; D000069284:Protein Translocation Systems; C477327:transporter associated with antigen processing (TAP); D000255:Adenosine Triphosphate