Nucleotide binding to the hydrophilic C-terminal domain of the transporter associated with antigen processing (TAP).
The gene products of tap1 and tap2 encoded in the major histocompatibility complex (MHC) class II region belong to the ATP binding cassette superfamily of transporters. They are thought to form a heterodimer for the delivery of peptides into the lumen of the endoplasmic reticulum; peptides are required for correct assembly and presentation of the MHC class I molecule peptide complex at the cell surface. To elucidate the ATP binding properties of these proteins in vitro, we expressed the hydrophilic C-terminal part of human transporter associated with antigen processing (TAP1) (nucleotide binding domain (NBD)-TAP1, amino acids 452-748) and TAP2 (NBD-TAP2, amino acids 399-686) fused to a His6 tag in Escherichia coli. The recombinant proteins accumulated exclusively in inclusion bodies and were solubilized under denaturing conditions. After purification by immobilized metal ion affinity chromatography, we were able to refold the domains for functional studies. NBD-TAP1 bound to C-8-ATP-agarose and was specifically eluted with ATP or EDTA. Photoaffinity labeling of NBD-TAP1 with the ATP analogues 8-azido-[gamma-32P]ATP and 3'-O-[(4-azido-3,5-[125I]diiodo-2-hydroxybenzoyl)-beta-alanyl]-ATP was specific. The addition of 50 microM ATP inhibited photoaffinity labeling by 8-azido-ATP down to 8% of controls. Efficiency of inhibition decreased as follows: ATP > GTP > ADP > CTP > AMP. Photolabeling of NBD-TAP2 was not observed. ATP hydrolysis by NBD-TAP1 was not detected. Until now strong but only indirect data of the TAP function existed. The described experiments demonstrate ATP binding to an isolated domain of the antigenic peptide transporter, TAP, and therefore support the theory of ATP-dependent peptide translocation.
Müller
K M
KM
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Ebensperger
C
C
Tampé
R
R
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000302:isolation & purification / Q000378:metabolism*; D000345:Affinity Labels; D017951:Antigen Presentation; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D017931:DNA Primers; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D002352:Carrier Proteins; D017931:DNA Primers; D000949:Histocompatibility Antigens Class II; C073119:TAP1 protein, human; C071692:TAP2 protein, human; D000255:Adenosine Triphosphate