ABCMdb

ABCB3 p.His661Ala

None has been submitted yet.

Publications

PMID: 17068338 [PubMed] Perria CL et al: "Catalytic site modifications of TAP1 and TAP2 and their functional consequences."

No. Sentence Comment

73 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment
74 The primers used were H661A forward, 5Ј-CTGGTGATTGCTGCCAGGCTGCAGACA-3Ј and H661A reverse, 5Ј-TGTCTGCAGCCTGGCAGCAATCA- CCAG-3Ј. Login to comment
195 As expected from labeling analyses with the single mutants, labeling of TAP1(D668N)⅐TAP2(E632Q) was more efficient than that of TAP1⅐TAP2 (data not shown), although because fluorescence protein-tagged versions of mutant TAP1 or TAP2 were not available, it was not possible to resolve labeling of the individual subunits. Login to comment
196 The TAP2(E632Q) and (E632Q/H661A) Mutations Significantly Impact the Ability of TAP2 to Induce MHC Class I Surface Expression in TAP2-deficient Cells, whereas Small Effects Are Observed with the Counterpart TAP1 Mutations (D668N and D668N/Q701A)-To extend the analyses of peptide translocation activity to assessments of the abilities of the TAP mutants to restore MHC class I surface expression in TAP-deficient human cells, retroviral constructs were generated that encoded TAP1, TAP1(D668N), TAP2, and TAP2(E632Q). Login to comment
208 The peptide translocation assays in insect cells, however, seemed to have lower sensitivity, as the low activity of the TAP2(E632Q) mutant complexes was more readily detectable using TAP activity assays that measured restoration of MHC class I surface expression. Login to comment
211 To additionally explore the effect of the switch region residues on TAP activity, we generated another retroviral construct encoding the TAP2(E632Q/H661A) double mutant, and assessed the ability of the mutant to restore MHC class I surface expression in TAP2-deficient cells. Login to comment
212 The mean fluorescence values of cells expressing TAP2(E632Q/H661A) ranged from 27 to 34% relative to that observed with cells expressing wild type TAP2 (six measurements), only slightly greater than that of the parent STF-1 cells that were TAP2-deficient (mean fluorescence 19-31% relative to wild type) (Fig. 4, C and G). Login to comment
213 In each of six- independent flow cytometric analyses, the TAP2(E632Q/ H661A) double mutant was more significantly impaired than the TAP2(E632Q) single mutant. Login to comment
214 However, in each of the analyses, residual enhancement of MHC class I surface expression was consistently observable in cells expressing the TAP2(E632Q/H661A) double mutant compared with unin- TABLE 1 Apparent affinities of indicated TAP constructs for 8-azido-͓␥-32 P͔ATP and 8-azido-͓␣-32 P͔ADP when expressed as single subunits or in complex with the indicated partner subunit The first four rows of measurements correspond to the derived apparent affinities of the wild type TAP1 or TAP1(D668N) for 8-azido-[␥-32 P]ATP and 8-azido-[␣- 32 P]ADP when expressed individually or in complex with TAP2-eYFP. Login to comment
227 The abbreviations are: WT for wild type TAP2, EQ for TAP2(E632Q), EQHA for TAP2(E632Q/H661A), ED for TAP2(E632D), HQ for TAP2(H661Q), and EDHQ for TAP2(E632D/H661Q). Login to comment
235 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39846 fected cells (representative analysis is shown Fig. 4C), indicating that the peptide translocation activity of TAP complexes was not completely impaired by the TAP2(E632Q/H661A) double mutation. Login to comment
241 The TAP2(E632D) and (H632Q) Mutations Result in Attenuated TAP Activity, whereas the TAP1(D668E/Q701H) Mutation Induces a Slight Increase in TAP Activity-In TAP1 sequences, Asp668 and Gln701 replace highly conserved glutamic acidandhistidineresidues,respectively,inotherABCtransporters (Fig. 1). Login to comment
242 Our results (Figs. 2 and 4) indicated that TAP1(D668N) andTAP1(D668N/Q701A)mutationsaffected TAP function less significantly than the counterpart TAP2(E632Q) and TAP2(E632Q/H661A) mutations. Login to comment
295 When the ATPase activity of the TAP2 site is reduced to a level below that of the TAP1 site (as might be the case with TAP2(E632Q/H661A)), it is possible that hydrolysis at the TAP1 site drives the residual transport. Login to comment
72 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment
210 To additionally explore the effect of the switch region residues on TAP activity, we generated another retroviral construct encoding the TAP2(E632Q/H661A) double mutant, and assessed the ability of the mutant to restore MHC class I surface expression in TAP2-deficient cells. Login to comment
226 The abbreviations are: WT for wild type TAP2, EQ for TAP2(E632Q), EQHA for TAP2(E632Q/H661A), ED for TAP2(E632D), HQ for TAP2(H661Q), and EDHQ for TAP2(E632D/H661Q). Login to comment
234 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39846 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 52ߦDECEMBER 29, 2006 fected cells (representative analysis is shown Fig. 4C), indicating that the peptide translocation activity of TAP complexes was not completely impaired by the TAP2(E632Q/H661A) double mutation. Login to comment
294 When the ATPase activity of the TAP2 site is reduced to a level below that of the TAP1 site (as might be the case with TAP2(E632Q/H661A)), it is possible that hydrolysis at the TAP1 site drives the residual transport. Login to comment
70 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment
71 The primers used were H661A forward, 5b18;-CTGGTGATTGCTGCCAGGCTGCAGACA-3b18; and H661A reverse, 5b18;-TGTCTGCAGCCTGGCAGCAATCA- CCAG-3b18;. Login to comment
193 The TAP2(E632Q) and (E632Q/H661A) Mutations Significantly Impact the Ability of TAP2 to Induce MHC Class I Surface Expression in TAP2-deficient Cells, whereas Small Effects Are Observed with the Counterpart TAP1 Mutations (D668N and D668N/Q701A)-To extend the analyses of peptide translocation activity to assessments of the abilities of the TAP mutants to restore MHC class I surface expression in TAP-deficient human cells, retroviral constructs were generated that encoded TAP1, TAP1(D668N), TAP2, and TAP2(E632Q). Login to comment
209 The mean fluorescence values of cells expressing TAP2(E632Q/H661A) ranged from 27 to 34% relative to that observed with cells expressing wild type TAP2 (six measurements), only slightly greater than that of the parent STF-1 cells that were TAP2-deficient (mean fluorescence 19-31% relative to wild type) (Fig. 4, C and G). Login to comment
224 The abbreviations are: WT for wild type TAP2, EQ for TAP2(E632Q), EQHA for TAP2(E632Q/H661A), ED for TAP2(E632D), HQ for TAP2(H661Q), and EDHQ for TAP2(E632D/H661Q). Login to comment
232 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39846 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 52ߦDECEMBER 29, 2006 fected cells (representative analysis is shown Fig. 4C), indicating that the peptide translocation activity of TAP complexes was not completely impaired by the TAP2(E632Q/H661A) double mutation. Login to comment
239 Our results (Figs. 2 and 4) indicated that TAP1(D668N) andTAP1(D668N/Q701A)mutationsaffected TAP function less significantly than the counterpart TAP2(E632Q) and TAP2(E632Q/H661A) mutations. Login to comment
292 When the ATPase activity of the TAP2 site is reduced to a level below that of the TAP1 site (as might be the case with TAP2(E632Q/H661A)), it is possible that hydrolysis at the TAP1 site drives the residual transport. Login to comment