ABCMdb

ABCB3 p.Glu632Gln

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

44 Site-directed mutations (D668N in TAP1; E632Q in TAP2) wereintro- duced into the TAP1-his and TAP2 constructs in the pPCR2.1 vector (17) using the QuikChange site-directed mutagenesis kit. Login to comment
73 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment
76 The primers used for TAP2(E632D) were E632D forward, 5Ј-CTCATCCTGGAT- GATGCTACTAGTGCC-308; and E632D reverse, 5Ј-GGCACTA- GTAGCATCATCCAGGATGAG-3Ј. Login to comment
78 TAP2(E632DH661Q) was generatedusingTAP2(E632D)asatemplateandusingtheprimers for TAP2(H661Q). Login to comment
79 Sequences encoding TAP2, TAP2(E632Q), TAP2(E632D), TAP2(H661Q), TAP2(E632D/H661Q), and TAP2(E632Q/ H662A) were excised from pPCR2.1 with BglII and XhoI and inserted into pMSCV 2.1 that was digested with BglII and XhoI. Login to comment
113 RESULTS The TAP2(E632Q) Mutation Impacts Peptide Translocation More Significantly Than the TAP1(D668N) Mutation-A histidine-tagged version of mutant TAP1(D668N) and untagged TAP2(E632Q) were expressed in insect cells along with the partner wild type subunits, using baculoviruses encoding the desired proteins. Login to comment
114 Microsomes were prepared of TAP1(D668N)⅐ TAP2, TAP1⅐TAP2(E632Q), or TAP1(D668N)⅐TAP2(E632Q) combinations, or wild type proteins, under conditions in which comparable levels of wild type or mutant proteins were expressed. Login to comment
117 Consistent with this expectation, a determination of KD values for binding of a fluorescent peptide substrate to TAP1(D668N)⅐TAP2 or TAP1⅐TAP2(E632Q) compared with ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites DECEMBER 29, 2006•VOLUME 281•NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 39841 the corresponding wild type complexes (TAP1-his⅐TAP2 and TAP1⅐TAP2, respectively) revealed no difference in calculated affinities (data not shown). Login to comment
129 The TAP1(D668N)⅐TAP2 complex is able to transport peptide (A), low to residual activity is observable with TAP1⅐TAP2(E632Q) (B), whereas the double mutant (C) did not display measurable activity by these assays. Login to comment
130 The WT microsomes used were TAP1⅐TAP2 for comparisons with TAP1⅐TAP2(E632Q), and TAP1-his⅐TAP2 for comparisons with TAP1(D668N)⅐TAP2) and TAP1(D668N)⅐TAP2(E632Q). Login to comment
136 DN indicates TAP1(D668N)⅐TAP2, EQ indicates TAP1⅐TAP2(E632Q), and DN.EQ indicates TAP1(D668N)⅐TAP2(E632Q). Login to comment
137 The graph indicates average ϩATP/-ATP ratios from four independent translocation experiments for TAP1(D668N)⅐TAP2, five independent experiments for TAP1⅐TAP2(E632Q), and four independent experiments for TAP1(D668N)⅐TAP2(E632Q) (containing five independent comparisons of expression matched wild type and mutant). Login to comment
142 For the TAP1⅐TAP2(E632Q), analyses, H0: WT ϭ M versus HA: WT Ͼ M, p value ϭ 0.0004465 (highly significant). Login to comment
143 Additionally, for TAP1⅐TAP2(E632Q), H0: C ϭ M versus HA: C Ͻ M, p value ϭ 0.01052 (significant). Login to comment
144 For the TAP1(D668N)⅐TAP2(E632Q) analyses, H0: WT ϭ M versus HA: WT Ͼ M, p value ϭ 0.002718 (highly significant). Login to comment
145 Additionally, for TAP1(D668N)⅐TAP2(E632Q), H0: C ϭ M versus HA: C Ͻ M: p value ϭ 0.07645 (not significant). Login to comment
151 For TAP1⅐ TAP2(E632Q), the e9;atp signals were only slightly higher compared with the -atp signals (Fig. 2B), whereas very similar ϩatp and -atp signals were observed with TAP1(D668N)!50; TAP2(E632Q). Login to comment
152 Fig. 2E shows the compiled average ϩatp/ -atp ratios from four independent peptide translocation experiments with TAP1(D668N)⅐TAP2, five independent experiments with TAP1⅐TAP2(E632Q), and four independent experiments with TAP1(D668N)⅐TAP2(E632Q). Login to comment
153 The compiled data in Fig. 2E, as well as the individual experiments shown in Fig. 2, A-C, indicated that the TAP1(D668N)⅐TAP2 combination was significantly translocation active, whereas TAP1⅐TAP2(E632Q) and TAP1(D668N)⅐TAP2(E632Q) complexes were significantly impaired relative to wild type TAP. Login to comment
154 Furthermore, the TAP1⅐TAP2(E632Q) mutant microsomes had slightly higher ϩatp/-atp ratios compared with control microsomes, indicating low but measurable activity. Login to comment
156 The TAP1(D668N) and TAP2(E632Q) Mutations Enhance Efficiencies of Labeling of TAP Complexes with 8-Azido-nucleotides-The E632Q and D668N mutations target putative ␥-phosphate contact residues; these mutations are not expected to alter nucleotide binding affinities. Login to comment
172 Based on several reports that the counterpart E632Q mutation of ABC transporter NBDs stabilizes nucleotide-bound NBD dimers (for example, Ref. 28), it is possible that enhanced labeling of TAP1(D668N) reflects the stabilization of NBD interactions both in the presence of 8-azido-ATP and 8-azido-ADP. Login to comment
176 TAP2 when expressed alone binds weakly to 8-azido-[␥- 32 P]ATP as previously described (18); the TAP2(E632Q) mutation did not enhance the 8-azido-ATP binding affinity. Login to comment
177 Low labeling signals were obtained for binding of both TAP2 and TAP2(E632Q) to 8-azido-[$25;-32 P]ATP, which were difficult to quantify (data not shown). Login to comment
178 When TAP2 or TAP2(E632Q) were expressed in combination with TAP1-eGFP, labeling of TAP2(E632Q) was more efficient than that of TAP2, in analyses with both 8-azido-ATP and 8-azido-ADP (Fig. 3, G and I). Login to comment
179 The TAP2(E632Q) mutation also enhanced the labeling efficiency ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites DECEMBER 29, 2006•VOLUME 281•NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 39843 FIGURE 3. Login to comment
180 Labeling with 8-azido-nucleotides of TAP complexes with a single TAP1(D668N) or TAP2(E632Q) mutation. Login to comment
186 F, immunoblotting analyses of microsomes expressing TAP1-eGFP⅐TAP2 (WT, lanes 1 and 3) or TAP1-eGFP⅐TAP2(E632Q)(M,lanes2and4).GandI,microsomesshowninlanes1and2ofFwereusedin8-azido-[␥-32 P]ATPlabelinganalysesandmicrosomesshown in lanes 3 and 4 of F were used in 8-azido-[␣-32 P]ADP labeling analyses. Login to comment
191 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39844 of TAP1-eGFP in complex (Fig. 3, G and I), to an extent greater than or equal to the enhancement of labeling of TAP2(E632Q) itself (Fig. 3, H and J). Login to comment
192 The observation that the TAP2(E632Q) mutation enhances 8-azido-ATP labeling of TAP1 residues (cross-labeling, Ref. 18) is consistent with the possibility that this mutation stabilizes nucleotide-bound conformations of both TAP1 and TAP2, such as would be observed in a transition-state TAP1⅐TAP2 NBD dimer. 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
197 TAP2-deficient STF-1 cells (25) were infected with the viruses encoding wild type TAP2 or TAP2(E632Q). Login to comment
199 Consistent with the marked reduction in the peptide translocation activity of TAP complexes that was induced by the TAP2(E632Q) mutation, the mutant had a significantly reduced ability to restore MHC class I surface expression in TAP2-deficient cells (Fig. 4A). Login to comment
200 Over 10 independent flow cytometric analyses, STF-1 cells infected with viruses encoding TAP2(E632Q) displayed an average mean fluorescence ratio of 42% relative to that observed with cells that were infected with wild type TAP2 (Fig. 4G). Login to comment
202 The average mean fluorescence ratio of the parent uninfected STF-1 cells was 23% relative to cells infected with the wild type TAP2-encoding virus; Fig. 4G), indicating low transport activity of TAP complexes containing TAP2(E632Q) (Fig. 4G). 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
216 Rows 7-10 correspond to the derived apparent affinities of the wild type TAP2 or TAP2(E632Q) for 8-azido-[␥-32 P]ATP and 8-azido-[␣-32 P]ADP when expressed individually or in complex with TAP1-eGFP. Login to comment
217 The last two rows of measurements correspond to the derived apparent affinities of TAP1-eGFP for 8-azido-[␥-32 P]ATP and 8-azido-[␣-32 P]ADP when expressed in complex with wild type TAP2 or TAP2(E632Q). Login to comment
220 The labeling efficiencies of single subunit TAP2 and TAP2(E632Q) with 8-azido-[␥-32 P]ATP were very inefficient and not accurately quantifiable (ND), as previously described for single subunit TAP2 (18). Login to comment
221 Subunit for which KD value is derived Partner subunit KD(ATP) KD(ADP) ␮M TAP1 None 2.5 1.8 Ϯ 0.6 TAP1(D668N) None 1.1 Ϯ 0.6 2.2 Ϯ 2.3 TAP1 TAP2-eYFP 2.6 Ϯ 1.5 0.7 Ϯ 0.5 TAP1(D668N) TAP2-eYFP 1.7 Ϯ 0.6 2.0 Ϯ 0.03 TAP2-eYFP TAP1 1.6 Ϯ 1.3 0.7 Ϯ 0.6 TAP2-eYFP TAP1(D668N) 2.7 Ϯ 0.6 2.2 Ϯ 1.0 TAP2 None NDa 2.3 Ϯ 1.1 TAP2(E632Q) None ND 4.9 Ϯ 0.1 TAP2 TAP1-eGFP 2.1 Ϯ 0.5 1.1 Ϯ 0.01 TAP2(E632Q) TAP1-eGFP 2.1 Ϯ 1.7 2.4 Ϯ 2.7 TAP1-eGFP TAP2 1.5 Ϯ 0.04 1.4 Ϯ 0.3 TAP1-eGFP TAP2(E632Q) 1.1 Ϯ 1.0 1.3 Ϯ 1.4 a ND, not determined. 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
286 Alternatively, blocking hydrolysis at a single site, such as with the TAP2(E632Q) mutant described here, could result in enhanced labeling of both TAP1 and TAP2, if the complexes are trapped in a dimeric conformation (Fig. 3J). 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
112 RESULTS The TAP2(E632Q) Mutation Impacts Peptide Translocation More Significantly Than the TAP1(D668N) Mutation-A histidine-tagged version of mutant TAP1(D668N) and untagged TAP2(E632Q) were expressed in insect cells along with the partner wild type subunits, using baculoviruses encoding the desired proteins. Login to comment
116 Consistent with this expectation, a determination of KD values for binding of a fluorescent peptide substrate to TAP1(D668N)ዼTAP2 or TAP1ዼTAP2(E632Q) compared with ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites DECEMBER 29, 2006ߦVOLUME 281ߦNUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 39841 at SEMMELWEIS UNIV OF MEDICINE on December 5, the corresponding wild type complexes (TAP1-hisዼTAP2 and TAP1ዼTAP2, respectively) revealed no difference in calculated affinities (data not shown). Login to comment
128 The TAP1(D668N)ዼTAP2 complex is able to transport peptide (A), low to residual activity is observable with TAP1ዼTAP2(E632Q) (B), whereas the double mutant (C) did not display measurable activity by these assays. Login to comment
135 DN indicates TAP1(D668N)ዼTAP2, EQ indicates TAP1ዼTAP2(E632Q), and DN.EQ indicates TAP1(D668N)ዼTAP2(E632Q). Login to comment
141 For the TAP1ዼTAP2(E632Q), analyses, H0: WT afd; M versus HA: WT b0e; M, p value afd; 0.0004465 (highly significant). Login to comment
150 For TAP1ዼ TAP2(E632Q), the af9;atp signals were only slightly higher compared with the afa;atp signals (Fig. 2B), whereas very similar af9;atp and afa;atp signals were observed with TAP1(D668N)ዼ TAP2(E632Q). Login to comment
155 The TAP1(D668N) and TAP2(E632Q) Mutations Enhance Efficiencies of Labeling of TAP Complexes with 8-Azido-nucleotides-The E632Q and D668N mutations target putative ॹ-phosphate contact residues; these mutations are not expected to alter nucleotide binding affinities. Login to comment
171 Based on several reports that the counterpart E632Q mutation of ABC transporter NBDs stabilizes nucleotide-bound NBD dimers (for example, Ref. 28), it is possible that enhanced labeling of TAP1(D668N) reflects the stabilization of NBD interactions both in the presence of 8-azido-ATP and 8-azido-ADP. Login to comment
175 TAP2 when expressed alone binds weakly to 8-azido-[ॹ- 32 P]ATP as previously described (18); the TAP2(E632Q) mutation did not enhance the 8-azido-ATP binding affinity. Login to comment
185 F, immunoblotting analyses of microsomes expressing TAP1-eGFPዼTAP2 (WT, lanes 1 and 3) or TAP1-eGFPዼTAP2(E632Q)(M,lanes2and4).GandI,microsomesshowninlanes1and2ofFwereusedin8-azido-[ॹ-32 P]ATPlabelinganalysesandmicrosomesshown in lanes 3 and 4 of F were used in 8-azido-[ॷ-32 P]ADP labeling analyses. Login to comment
190 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39844 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 52ߦDECEMBER 29, 2006 of TAP1-eGFP in complex (Fig. 3, G and I), to an extent greater than or equal to the enhancement of labeling of TAP2(E632Q) itself (Fig. 3, H and J). Login to comment
194 As expected from labeling analyses with the single mutants, labeling of TAP1(D668N)ዼTAP2(E632Q) was more efficient than that of TAP1ዼTAP2 (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
198 Consistent with the marked reduction in the peptide translocation activity of TAP complexes that was induced by the TAP2(E632Q) mutation, the mutant had a significantly reduced ability to restore MHC class I surface expression in TAP2-deficient cells (Fig. 4A). Login to comment
201 The average mean fluorescence ratio of the parent uninfected STF-1 cells was 23% relative to cells infected with the wild type TAP2-encoding virus; Fig. 4G), indicating low transport activity of TAP complexes containing TAP2(E632Q) (Fig. 4G). Login to comment
207 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
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
215 Rows 7-10 correspond to the derived apparent affinities of the wild type TAP2 or TAP2(E632Q) for 8-azido-[ॹ-32 P]ATP and 8-azido-[ॷ-32 P]ADP when expressed individually or in complex with TAP1-eGFP. Login to comment
219 The labeling efficiencies of single subunit TAP2 and TAP2(E632Q) with 8-azido-[ॹ-32 P]ATP were very inefficient and not accurately quantifiable (ND), as previously described for single subunit TAP2 (18). 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
285 Alternatively, blocking hydrolysis at a single site, such as with the TAP2(E632Q) mutant described here, could result in enhanced labeling of both TAP1 and TAP2, if the complexes are trapped in a dimeric conformation (Fig. 3J). 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
42 Site-directed mutations (D668N in TAP1; E632Q in TAP2) wereintro- duced into the TAP1-his and TAP2 constructs in the pPCR2.1 vector (17) using the QuikChange site-directed mutagenesis kit. For TAP1, the primers were: 5b18;-GTGTACTTATTCTAGATAA- TGCCACCAGTGCCCTG-3b18; and 5b18;-CAGGGCACTGGTGGC- ATTATCTAGAATAAGTACAC-3b18; (reverse primer). Login to comment
70 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment
110 RESULTS The TAP2(E632Q) Mutation Impacts Peptide Translocation More Significantly Than the TAP1(D668N) Mutation-A histidine-tagged version of mutant TAP1(D668N) and untagged TAP2(E632Q) were expressed in insect cells along with the partner wild type subunits, using baculoviruses encoding the desired proteins. Login to comment
111 Microsomes were prepared of TAP1(D668N)ዼ TAP2, TAP1ዼTAP2(E632Q), or TAP1(D668N)ዼTAP2(E632Q) combinations, or wild type proteins, under conditions in which comparable levels of wild type or mutant proteins were expressed. Login to comment
126 The TAP1(D668N)ዼTAP2 complex is able to transport peptide (A), low to residual activity is observable with TAP1ዼTAP2(E632Q) (B), whereas the double mutant (C) did not display measurable activity by these assays. Login to comment
127 The WT microsomes used were TAP1ዼTAP2 for comparisons with TAP1ዼTAP2(E632Q), and TAP1-hisዼTAP2 for comparisons with TAP1(D668N)ዼTAP2) and TAP1(D668N)ዼTAP2(E632Q). Login to comment
133 DN indicates TAP1(D668N)ዼTAP2, EQ indicates TAP1ዼTAP2(E632Q), and DN.EQ indicates TAP1(D668N)ዼTAP2(E632Q). Login to comment
134 The graph indicates average af9;ATP/afa;ATP ratios from four independent translocation experiments for TAP1(D668N)ዼTAP2, five independent experiments for TAP1ዼTAP2(E632Q), and four independent experiments for TAP1(D668N)ዼTAP2(E632Q) (containing five independent comparisons of expression matched wild type and mutant). Login to comment
139 For the TAP1ዼTAP2(E632Q), analyses, H0: WT afd; M versus HA: WT b0e; M, p value afd; 0.0004465 (highly significant). Login to comment
140 Additionally, for TAP1ዼTAP2(E632Q), H0: C afd; M versus HA: C b0d; M, p value afd; 0.01052 (significant). Login to comment
148 For TAP1ዼ TAP2(E632Q), the af9;atp signals were only slightly higher compared with the afa;atp signals (Fig. 2B), whereas very similar af9;atp and afa;atp signals were observed with TAP1(D668N)ዼ TAP2(E632Q). Login to comment
149 Fig. 2E shows the compiled average af9;atp/ afa;atp ratios from four independent peptide translocation experiments with TAP1(D668N)ዼTAP2, five independent experiments with TAP1ዼTAP2(E632Q), and four independent experiments with TAP1(D668N)ዼTAP2(E632Q). Login to comment
169 Based on several reports that the counterpart E632Q mutation of ABC transporter NBDs stabilizes nucleotide-bound NBD dimers (for example, Ref. 28), it is possible that enhanced labeling of TAP1(D668N) reflects the stabilization of NBD interactions both in the presence of 8-azido-ATP and 8-azido-ADP. Login to comment
173 TAP2 when expressed alone binds weakly to 8-azido-[ॹ- 32 P]ATP as previously described (18); the TAP2(E632Q) mutation did not enhance the 8-azido-ATP binding affinity. Login to comment
174 Low labeling signals were obtained for binding of both TAP2 and TAP2(E632Q) to 8-azido-[ॹ-32 P]ATP, which were difficult to quantify (data not shown). Login to comment
183 F, immunoblotting analyses of microsomes expressing TAP1-eGFPዼTAP2 (WT, lanes 1 and 3) or TAP1-eGFPዼTAP2(E632Q)(M,lanes2and4).GandI,microsomesshowninlanes1and2ofFwereusedin8-azido-[ॹ-32 P]ATPlabelinganalysesandmicrosomesshown in lanes 3 and 4 of F were used in 8-azido-[ॷ-32 P]ADP labeling analyses. Login to comment
188 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39844 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 52ߦDECEMBER 29, 2006 of TAP1-eGFP in complex (Fig. 3, G and I), to an extent greater than or equal to the enhancement of labeling of TAP2(E632Q) itself (Fig. 3, H and J). Login to comment
189 The observation that the TAP2(E632Q) mutation enhances 8-azido-ATP labeling of TAP1 residues (cross-labeling, Ref. 18) is consistent with the possibility that this mutation stabilizes nucleotide-bound conformations of both TAP1 and TAP2, such as would be observed in a transition-state TAP1ዼTAP2 NBD dimer. 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
205 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
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
218 Subunit for which KD value is derived Partner subunit KD(ATP) KD(ADP) òe;M TAP1 None 2.5 1.8 afe; 0.6 TAP1(D668N) None 1.1 afe; 0.6 2.2 afe; 2.3 TAP1 TAP2-eYFP 2.6 afe; 1.5 0.7 afe; 0.5 TAP1(D668N) TAP2-eYFP 1.7 afe; 0.6 2.0 afe; 0.03 TAP2-eYFP TAP1 1.6 afe; 1.3 0.7 afe; 0.6 TAP2-eYFP TAP1(D668N) 2.7 afe; 0.6 2.2 afe; 1.0 TAP2 None NDa 2.3 afe; 1.1 TAP2(E632Q) None ND 4.9 afe; 0.1 TAP2 TAP1-eGFP 2.1 afe; 0.5 1.1 afe; 0.01 TAP2(E632Q) TAP1-eGFP 2.1 afe; 1.7 2.4 afe; 2.7 TAP1-eGFP TAP2 1.5 afe; 0.04 1.4 afe; 0.3 TAP1-eGFP TAP2(E632Q) 1.1 afe; 1.0 1.3 afe; 1.4 a ND, not determined. 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
283 Alternatively, blocking hydrolysis at a single site, such as with the TAP2(E632Q) mutant described here, could result in enhanced labeling of both TAP1 and TAP2, if the complexes are trapped in a dimeric conformation (Fig. 3J). 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

PMID: 24196954 [PubMed] Geng J et al: "Analyses of conformational states of the transporter associated with antigen processing (TAP) protein in a native cellular membrane environment."

No. Sentence Comment

41 The hydrolysis-deficient TAP2 mutant TAP2-EYFP(E632Q) was made by introducing the E632Q mutation into TAP2-EYFP using the QuikChange site-directed mutagenesis kit. Login to comment
77 A fluorophore-labeled TAP substrate (RYWANATKFITCSR) was incubated with control cells, cells expressing wild-type TAP complexes, T1C-T2Y, or an ATPase-deficient mutant complex, T1C-T2Y(E632Q) (11). Login to comment
78 The glutamate-to-glutamine mutation at the residue C-terminal to the Walker B motif, as in TAP2(E632Q), impairs ATP hydrolysis and NBD dimer separation in many ABC transporters (8, 16, 17). Login to comment
82 The ATPase-deficient mutant complex T1C/T2Y(E632Q) did not transport peptides, as expected from previous measurements with untagged mutant complexes (11) (Fig. 2B). Login to comment
114 As can be seen in Fig. 3, C and D, with the ATPase-deficient TAP mutant complex T1C/T2Y(E632Q), the addition of ATP, but not ADP, markedly increased the FRET signal even at 4 &#b0;C (Fig. 3C). Login to comment
168 We expected that the ATPase-deficient mutant TAP1-TAP2(E632Q) would also arrest the catalytic cycle of TAP in a prehydrolysis reaction intermediate in the presence of both ATP and peptide because TAP1-TAP2(E632Q) complexes are able to bind nucleotides and peptide with similar affinities as wild-type TAP (11). Login to comment
169 As shown in Fig. 2B, TAP1-TAP2(E632Q) complexes were impaired for peptide transport into sf9 cell microsomes because of a deduced deficiency in ATPase activity. Login to comment
172 At 4 &#b0;C (Fig. 5G), similar FRET efficiencies were observed with T1C-T2Y(E632Q) complexes in the presence of both nucleotides and peptide, compared with that observed in the presence of peptide alone. Login to comment
190 B-H, FRET efficiency changes were measured in permeabilized cells expressing the T1C-T2Y or T1C-T2Y(E632Q) complexes in the presence of nucleotide alone, peptide RKL alone, nucleotide af9; the peptide RKL, or, additionally, in the presence of vanadate (Vi) at 4 &#b0;C or 27 &#b0;C as indicated. Login to comment
197 The T1C-T2Y(E632Q) complex is more sensitive to nucleotides than the wild-type TAP complex, as suggested by the findings of ATP-induced enhancement in FRET efficiency, even at 4 &#b0;C (Fig. 3C), and a more significant enhancement in FRET at 27 &#b0;C compared with wild-type complexes (normalized FRET efficiency changes of b03;3.6 and b03;2%, respectively). Login to comment
198 These findings are consistent with our previous study, which showed that, compared with wild-type TAP1-TAP2, the TAP2(E632Q) mutation enhances labeling of both TAP1 and TAP2 with 8-azido-ATP (11). Login to comment
200 We found that nucleotide-induced TAP conformational change is temperature-dependent, indicated by higher FRET efficiencies of both the wild-type and T1C-T2Y(E632Q) mutant at 27 &#b0;C compared with 4 &#b0;C in the presence of nucleotide, both in the presence and in the absence of peptide. Login to comment
229 Interestingly, with T1C-T2Y(E632Q), ADP binding triggers NBD dimerization to the same extent as ATP in the presence of peptide. Login to comment
230 These findings are consistent with the possibility that the E632Q mutation of TAP2 lowers the energy barrier for the conformational change so that either ADP or ATP suffices to induce full closure. Login to comment