Head-head/tail-tail relative orientation of the pore-forming domains of the heterodimeric ABC transporter TAP.
BACKGROUND
The transporter associated with antigen processing (TAP) is a heterodimeric member of the large family of ABC transporters. The study of interactions between the subunits TAP1 and TAP2 can reveal the relative orientation of the transmembrane segments, which form a translocation pore for peptides. This is essential for understanding the architecture of TAP and other ABC transporters.


RESULTS
The amino-terminal six transmembrane segments (TMs) of human TAP1, TAP1 (1-6), and the amino-terminal five TMs of TAP2, TAP2(1-5), are thought to constitute the pore of TAP. Two new approaches are used to define dimer interactions. We show that TM6 of TAP1 (1-6) is able to change topology post-translationally. This TM, along with a cytoplasmic tail, is translocated into the endoplasmic reticulum lumen, unless TAP2 is expressed. Coexpression of TM(4-5) of TAP2 stabilizes the topology of TAP1 (1-6), even when the TM1 of TAP1 is subsitituted with another sequence. This suggests that the carboxy-terminal TMs of the pore-forming domains TAP1 (1-6) and TAP2(1-5) interact. An alternative assay uses photobleaching in living cells using TAP1 (1-6) tagged with the green fluorescent protein (GFP). Coexpression with TAP2(1-5) results in reduced movement of the heterodimer within the endoplasmic reticulum membrane, as compared with the single TAP1 (1-6) molecule. In contrast, TAP2(1-4) has no effect on the mobility of TAP1 (1-6)-GFP, indicating the importance of TM5 of TAP2 for dimer formation. Also, TM1 of both TAP1 and TAP2 is essential for formation of a complex with low mobility.


CONCLUSIONS
Dimerization of the pore-forming transmembrane domains of TAP1 (TM1-6) with its TAP2 counterpart (TM1-5) prevents the post-translational translocation of TM6 of TAP1 and results in a complex with reduced mobility within the endoplasmic reticulum membrane compared with the free subunit. These techniques are used to show that the pore-forming domains of TAP are aligned in a head-head/tail-tail orientation. This positions the following peptide-binding segments of the two TAP subunits to one side of the pore.
Vos
J C
JC
Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam.
Reits
E A
EA
Wojcik-Jacobs
E
E
Neefjes
J
J
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D046911:Macromolecular Substances; D010777:Photochemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; C073119:TAP1 protein, human; C071692:TAP2 protein, human