ABCMdb

PMID: 17068338

Perria CL, Rajamanickam V, Lapinski PE, Raghavan M
Catalytic site modifications of TAP1 and TAP2 and their functional consequences.
J Biol Chem. 2006 Dec 29;281(52):39839-51. Epub 2006 Oct 26., [PubMed]

Sentences

No. Mutations Sentence Comment

42 ABCB3 p.Glu632Gln 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 44 ABCB3 p.Glu632Gln 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 70 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment 71 ABCB3 p.His661Ala ABCB3 p.His661Ala The primers used were H661A forward, 5b18;-CTGGTGATTGCTGCCAGGCTGCAGACA-3b18; and H661A reverse, 5b18;-TGTCTGCAGCCTGGCAGCAATCA- CCAG-3b18;. Login to comment 72 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.Glu632Asp ABCB3 p.His661Gln The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment 73 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.His661Ala ABCB3 p.His661Ala ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment 74 ABCB3 p.His661Ala ABCB3 p.His661Ala ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln ABCB3 p.His661Gln The primers used were H661A forward, 5@8;-CTGGTGATTGCTGCCAGGCTGCAGACA-3Ј and H661A reverse, 5Ј-TGTCTGCAGCCTGGCAGCAATCA- CCAG-3Ј. Login to comment 75 ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln TAP2(E632D) and TAP2(H661Q) were generated using TAP2 in pPCR2.1 vector as the template. Login to comment 76 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln ABCB3 p.His661Gln ABCB3 p.His661Gln The primers used for TAP2(E632D) were E632D forward, 5Ј-CTCATCCTGGAT- GATGCTACTAGTGCC-308; and E632D reverse, 5Ј-GGCACTA- GTAGCATCATCCAGGATGAG-3Ј. Login to comment 77 ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln ABCB3 p.His661Gln The primers used for TAP2(H661Q) were H661Q forward, 5Ј-CTGGTGATTGCTC- AAAGGCTGCAGACA-3ЈandH661Qreverse,5Ј-TGTCTGCA- GCCTTTGAGCAATCACCAG-3Ј. Login to comment 78 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln ABCB3 p.His661Gln TAP2(E632DH661Q) was generatedusingTAP2(E632D)asatemplateandusingtheprimers for TAP2(H661Q). Login to comment 79 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln 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 110 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 112 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 113 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 116 ABCB3 p.Glu632Gln 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 117 ABCB3 p.Glu632Gln 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 126 ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 128 ABCB3 p.Glu632Gln 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 129 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 133 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln DN indicates TAP1(D668N)ዼTAP2, EQ indicates TAP1ዼTAP2(E632Q), and DN.EQ indicates TAP1(D668N)ዼTAP2(E632Q). Login to comment 134 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 135 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln DN indicates TAP1(D668N)ዼTAP2, EQ indicates TAP1ዼTAP2(E632Q), and DN.EQ indicates TAP1(D668N)ዼTAP2(E632Q). Login to comment 136 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln DN indicates TAP1(D668N)⅐TAP2, EQ indicates TAP1⅐TAP2(E632Q), and DN.EQ indicates TAP1(D668N)⅐TAP2(E632Q). Login to comment 137 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 139 ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln Additionally, for TAP1ዼTAP2(E632Q), H0: C afd; M versus HA: C b0d; M, p value afd; 0.01052 (significant). Login to comment 141 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln For the TAP1ዼTAP2(E632Q), analyses, H0: WT afd; M versus HA: WT b0e; M, p value afd; 0.0004465 (highly significant). Login to comment 142 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln Additionally, for TAP1⅐TAP2(E632Q), H0: C ϭ M versus HA: C Ͻ M, p value ϭ 0.01052 (significant). Login to comment 144 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln 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 148 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 150 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 151 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln Furthermore, the TAP1⅐TAP2(E632Q) mutant microsomes had slightly higher ϩatp/-atp ratios compared with control microsomes, indicating low but measurable activity. Login to comment 155 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 156 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 169 ABCB3 p.Glu632Gln 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 171 ABCB3 p.Glu632Gln 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 172 ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln 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 175 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 176 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln Labeling with 8-azido-nucleotides of TAP complexes with a single TAP1(D668N) or TAP2(E632Q) mutation. Login to comment 183 ABCB3 p.Glu632Gln 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 185 ABCB3 p.Glu632Gln 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 186 ABCB3 p.Glu632Gln 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 188 ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln 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 190 ABCB3 p.Glu632Gln 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 191 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 193 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 194 ABCB3 p.Glu632Gln 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 195 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln TAP2-deficient STF-1 cells (25) were infected with the viruses encoding wild type TAP2 or TAP2(E632Q). Login to comment 198 ABCB3 p.Glu632Gln 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 199 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln 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 201 ABCB3 p.Glu632Gln 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 202 ABCB3 p.Glu632Gln 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 205 ABCB3 p.Glu632Gln 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 207 ABCB3 p.Glu632Gln 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 208 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 210 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.His661Ala 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 211 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.His661Ala ABCB3 p.His661Ala 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.His661Ala 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.His661Ala 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 215 ABCB3 p.Glu632Gln 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 216 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 218 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 219 ABCB3 p.Glu632Gln 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 220 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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 224 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln 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 226 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln 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 227 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln 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 ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 234 ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 235 ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 238 ABCB3 p.Glu632Asp 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 239 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 240 ABCB3 p.Glu632Asp 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 241 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln 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 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 243 ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln ABCB3 p.His661Gln To further examine this possibility, we analyzed the effects of TAP2(E632D), TAP2(H661Q), and TAP2(E632D/H661Q) mutants upon TAP activity in STF-1 cells. Login to comment 244 ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln ABCB3 p.His661Gln To further examine this possibility, we analyzed the effects of TAP2(E632D), TAP2(H661Q), and TAP2(E632D/H661Q) mutants upon TAP activity in STF-1 cells. Login to comment 245 ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln The TAP2(E632D) mutation (41% activity relative to wild type) affected TAP activity more significantly than the TAP2(H661Q) mutation (63% relative to wild type), and the double mutant (39% relative to wild type) exhibited a similar level of impairment as the TAP2(E632D) mutation. Login to comment 246 ABCB3 p.Glu632Asp ABCB3 p.Glu632Asp ABCB3 p.His661Gln The TAP2(E632D) mutation (41% activity relative to wild type) affected TAP activity more significantly than the TAP2(H661Q) mutation (63% relative to wild type), and the double mutant (39% relative to wild type) exhibited a similar level of impairment as the TAP2(E632D) mutation. Login to comment 261 ABCB3 p.Glu632Asp ABCB3 p.His661Gln DISCUSSION Various modifications of TAP2 at Glu632 and His661 , including E632D and H661Q, significantly reduced the catalytic activity of TAP complexes (Figs. 2 and 4, A, C, E, and G). Login to comment 263 ABCB3 p.Glu632Asp ABCB3 p.His661Gln DISCUSSION Various modifications of TAP2 at Glu632 and His661 , including E632D and H661Q, significantly reduced the catalytic activity of TAP complexes (Figs. 2 and 4, A, C, E, and G). Login to comment 264 ABCB3 p.Glu632Asp ABCB3 p.His661Gln DISCUSSION Various modifications of TAP2 at Glu632 and His661 , including E632D and H661Q, significantly reduced the catalytic activity of TAP complexes (Figs. 2 and 4, A, C, E, and G). Login to comment 276 ABCB3 p.Glu632Asp ABCB3 p.His661Gln These data indicated a more important role for the switch region histidine in mediating ATP hydrolysis within isolated TAP1 NBD; however, at the TAP2 site of full-length TAP complexes, it appears that the TAP2(E632D) mutation causes a slightly greater loss in TAP activity compared with the TAP2(H661Q) mutation (Fig. 4E). Login to comment 278 ABCB3 p.Glu632Asp ABCB3 p.His661Gln These data indicated a more important role for the switch region histidine in mediating ATP hydrolysis within isolated TAP1 NBD; however, at the TAP2 site of full-length TAP complexes, it appears that the TAP2(E632D) mutation causes a slightly greater loss in TAP activity compared with the TAP2(H661Q) mutation (Fig. 4E). Login to comment 279 ABCB3 p.Glu632Asp ABCB3 p.His661Gln These data indicated a more important role for the switch region histidine in mediating ATP hydrolysis within isolated TAP1 NBD; however, at the TAP2 site of full-length TAP complexes, it appears that the TAP2(E632D) mutation causes a slightly greater loss in TAP activity compared with the TAP2(H661Q) mutation (Fig. 4E). Login to comment 283 ABCB3 p.Glu632Gln 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 285 ABCB3 p.Glu632Gln 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 286 ABCB3 p.Glu632Gln 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 287 ABCB3 p.Glu632Asp ABCB3 p.His661Gln Because the chimeric TAP1ዼT2MT1C complexes contained non-consensus residues (Asp668 and Gln701 ) at both nucleotide binding sites, the naturally occurring Glu b0e; Asp and His b0e; Gln alterations at these residues apparently did not completely abrogate the ability of the TAP1 site to hydrolyze ATP (consistent with the low but measurable activity of TAP2(E632D/H661Q); Fig. 4, E and G). Login to comment 289 ABCB3 p.Glu632Asp ABCB3 p.His661Gln Because the chimeric TAP1ዼT2MT1C complexes contained non-consensus residues (Asp668 and Gln701 ) at both nucleotide binding sites, the naturally occurring Glu b0e; Asp and His b0e; Gln alterations at these residues apparently did not completely abrogate the ability of the TAP1 site to hydrolyze ATP (consistent with the low but measurable activity of TAP2(E632D/H661Q); Fig. 4, E and G). Login to comment 290 ABCB3 p.Glu632Asp ABCB3 p.His661Gln Because the chimeric TAP1⅐T2MT1C complexes contained non-consensus residues (Asp668 and Gln701 ) at both nucleotide binding sites, the naturally occurring Glu Ͼ Asp and His Ͼ Gln alterations at these residues apparently did not completely abrogate the ability of the TAP1 site to hydrolyze ATP (consistent with the low but measurable activity of TAP2(E632D/H661Q); Fig. 4, E and G). Login to comment 292 ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 294 ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 295 ABCB3 p.Glu632Gln ABCB3 p.His661Ala 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 310 ABCB2 p.Glu3Ser ABCB2 p.Glu3Asp These include ABCC1-6 and ABCC10-11 (multidrug resistance proteins), ABCC8 and ABCC9 (SUR1 and SUR2) that all contain Walker B Glu 3 Asp substitutions in their NBD1, and NBD1 of ABCC7 (cystic fibrosis transmembrane conductance regulator, which has a Walker B Glu 3 Ser substitution). Login to comment 312 ABCB2 p.Glu3Ser ABCB2 p.Glu3Asp These include ABCC1-6 and ABCC10-11 (multidrug resistance proteins), ABCC8 and ABCC9 (SUR1 and SUR2) that all contain Walker B Glu 3 Asp substitutions in their NBD1, and NBD1 of ABCC7 (cystic fibrosis transmembrane conductance regulator, which has a Walker B Glu 3 Ser substitution). Login to comment 313 ABCB2 p.Glu3Ser ABCB2 p.Glu3Asp These include ABCC1-6 and ABCC10-11 (multidrug resistance proteins), ABCC8 and ABCC9 (SUR1 and SUR2) that all contain Walker B Glu 3 Asp substitutions in their NBD1, and NBD1 of ABCC7 (cystic fibrosis transmembrane conductance regulator, which has a Walker B Glu 3 Ser substitution). Login to comment