ABCMdb

ABCB3 p.Ala609Gly

Predicted by SNAP2:	C: D (53%), D: D (80%), E: D (59%), F: D (71%), G: N (78%), H: D (66%), I: D (63%), K: N (78%), L: D (71%), M: D (63%), N: D (66%), P: D (59%), Q: D (66%), R: D (71%), S: N (82%), T: N (82%), V: N (87%), W: D (75%), Y: D (71%),
Predicted by PROVEAN:	C: N, D: D, E: N, F: D, G: N, H: D, I: D, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D,

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Publications
[hide] Functional non-equivalence of ATP-binding cassette... J Biol Chem. 2004 Oct 29;279(44):46073-81. Epub 2004 Aug 17. Chen M, Abele R, Tampe R
Functional non-equivalence of ATP-binding cassette signature motifs in the transporter associated with antigen processing (TAP).
J Biol Chem. 2004 Oct 29;279(44):46073-81. Epub 2004 Aug 17., [PMID:15322097]

Abstract [show]
The transporter associated with antigen processing (TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine (LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.

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Sentences [show]
No. Sentence Comment
62 Site-directed mutations (L643A, TAP1; G646V, TAP1; G646D, TAP1; L607A, TAP2; G610V, TAP2; G610D, TAP2; S644A/G645A, TAP1; and A608S/A609G, TAP2) were introduced by sequence overlapping PCR using pFastBacDual.TAP1wt or pFastBacDual.TAP2wt as templates. Login to comment
63 The sequences of the sense oligonucleotides were as follows: 5Ј-GCTGGGAGCCAGGCG- TCAGGGGGTCAG-3Ј for TAP1L643A; 5Ј-CAGCTGTCAGGGGTTCAG- CGACAGGCAG-3Ј for TAP1G646V; 5Ј-CAGCTGTCAGGGGATCAGC- GACAGGCAG-3Ј for TAP1G646D; 5Ј-GGAAGCCAGGCGGCTGCGGG- ACAGAAAC-3Ј for TAP2L607A; 5Ј-GCTGGCTGCGGTACAGAAACAA- CGTC-3Ј for TAP2G610V; 5Ј-GCTGGCTGCGGACCAGAAACAACGTC- TG-3Ј for TAP2G610D; 5Ј-GCTGGGAGCCAGCTGGCAGCGGGTCAG- CGACAGGCA-3Ј for TAP1S644A/G645A; and 5Ј-GAAGGGAAGCCAG- CTGTCTGGGGGACAGAAACAACG-3Ј for TAP2A608S/A609G, respectively (mutated bases are underlined). Login to comment