Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle.
BACKGROUND
The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated.


RESULTS
Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2.


CONCLUSIONS
Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.
Alberts
P
P
Institute for Genetics, University of Cologne, D-50674, Cologne, Germany.
Daumke
O
O
Deverson
E V
EV
Howard
J C
JC
Knittler
M R
MR
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D006801:Humans; D008285:Major Histocompatibility Complex; D016296:Mutagenesis; D010455:Peptides / Q000378:metabolism
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D010455:Peptides; C073119:TAP1 protein, human; C071692:TAP2 protein, human; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate