ABCMdb

ABCB3 p.Lys509Met

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (91%), I: D (91%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Nucleotide interactions with membrane-bound transp... J Biol Chem. 2003 Mar 7;278(10):8229-37. Epub 2002 Dec 25. Lapinski PE, Raghuraman G, Raghavan M
Nucleotide interactions with membrane-bound transporter associated with antigen processing proteins.
J Biol Chem. 2003 Mar 7;278(10):8229-37. Epub 2002 Dec 25., [PMID:12501238]

Abstract [show]
The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4 degrees C using the photo-cross-linkable nucleotide analogs 8-azido-[gamma-(32)P]ATP and 8-azido-[alpha-(32)P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.

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46 EXPERIMENTAL PROCEDURES Baculoviruses for Expression of TAP1, TAP2, TAP1(K544M), TAP2(K509M), T2MT1C, T1MT2C, TAP1-eGFP, and TAP2-eYFP- Baculoviruses encoding wild type human TAP1 and TAP2 were obtained from the laboratory of Dr. Robert Tampe´ (22). Login to comment
47 We have previously described the construction of baculoviruses encoding the TAP1 mutant (TAP1(K544M) and the TAP2 mutant (TAP2(K509M)) (5). Login to comment
99 Mutation of the TAP2 Walker A lysine residue (TAP2(K509M)) reduced the TAP2-associated signal and derived affinity (KD Ͼ 20 ␮M when expressed in complex with TAP1-eGFP) (Fig. 2, E, bottom panel compared with top panel, and H and Table I). Login to comment
116 In the corresponding TAP1-eGFP/TAP2(K509M) complexes, a similar affinity was derived corresponding to TAP1-eGFP labeling (KD ϭ 2.8 Ϯ 2.9 ␮M) (Fig. 2I and Table I). Login to comment
118 However, the signals derived for TAP1-eGFP in the TAP1-eGFP/TAP2(K509M) mutant complex are reduced compared with that derived for the wild type complex (Fig. 2I), even though slightly higher levels of TAP1-eGFP were present in the mutant complex (Fig. 2C, lane 1 compared with lane 2). Login to comment
124 Insect cell microsomal membranes expressing TAP1 alone or TAP2-eYFP alone (A), the TAP1-eGFP/TAP2 or TAP1-eGFP/TAP2(K509M) combinations with the TAP1-eGFP component in excess (B), or the TAP1-eGFP/TAP2 or TAP1-eGFP/TAP2(K509M) combinations with the TAP2 or TAP2(K509M) components in excess (C) were incubated with different concentration of 8-azido-[␥-32 P]ATP for 15 min on ice and subsequently cross-linked by UV irradiation. Login to comment
133 Mutation of the TAP2 Walker A lysine TAP2(K509M) reduced TAP2 labeling and the corresponding affinity. Login to comment
135 The binding affinity corresponding to TAP1-eGFP labeling when in complex with wild type TAP2 is similar to that derived when TAP1 is in complex with the nucleotide binding-deficient TAP2(K509M). Login to comment
137 KD (ATP) n KD (ADP) n ␮M ␮M TAP2 nucleotide binding TAP2-eYFP 19.3 Ϯ 2.5 2 4.4 Ϯ 1.4 2 TAP2/TAP1-eGFP 2.7 Ϯ 1.0 3 0.5 Ϯ 0.1 2 TAP2(K509M)/TAP1-eGFP Ͼ20 4 9.0 Ϯ 1.4 2 TAP1 nucleotide binding TAP1 4.6 Ϯ 1.9 3 1.4 Ϯ 0.1 2 TAP1-eGFP/TAP2 2.1 Ϯ 0.8 3 0.7 Ϯ 0.1 3 TAP1-eGFP/TAP2(K509M) 2.8 Ϯ 2.9 4 0.6 Ϯ 0.2 3 The data shown in Fig. 2 were derived from analyses of 8-azido-[␥-32 P]ATP binding to TAP1/TAP2 complexes. Login to comment
161 For these analyses, we prepared microsomes containing TAP1(K544M)/ TAP2 complexes and TAP1(K544M)/TAP2(K509M) complexes. Login to comment
163 At comparable expression levels of both components (Fig. 4D), strong labeling was visualized for the TAP1(K544M)/TAP2 combination, whereas signals for the TAP1(K544M)/TAP2(K509M) combination were barely detectable (Fig. 4E, top and middle panels, respectively). Login to comment
173 eYFP complexes (Fig. 4E, bottom panel), a corresponding distinct signal was not visualized in the TAP1(K544M)/ TAP2(K509M) combination (Fig. 4E, middle panel), despite the higher expression of TAP1(K544M) in the latter complexes. Login to comment
184 Using TAP1-eGFP/TAP2(K509M) complexes under conditions of TAP1 excess or TAP2 excess (Fig. 2), we determined that the two nucleotide-binding sites of TAP1/TAP2 complexes did in fact bind 8-azido-ATP with apparent affinities that were, within the error of these measurements, quite similar to each other. Login to comment
199 E, phosphorimaging analyses of 8-azido-[␥-32 P]ATP binding to microsomes containing TAP1(K544M)/TAP2 (top panel), TAP1(K544M)/TAP2(K509M) (middle panel), or TAP1(K544M)/TAP2-eYFP (bottom panel). Login to comment
200 Signals corresponding to TAP1 (K544M) could be observed when in complex with TAP2-eYFP but not when in complex with TAP2(K509M). Login to comment
201 The absence of a signal was not due to the expression level, as TAP1(K544M) was expressed at higher levels in the microsomes with the TAP2(K509M) combination compared with the TAP2-eYFP combination (see D). Login to comment
209 Mutation of the TAP2 Walker A lysine residue (TAP2(K509M)) indeed influenced nucleotide binding at the TAP2 site (Fig. 2). Login to comment
214 However, co-expression of TAP1(K544M) with TAP2(K509M) resulted in a nucleotide-binding deficient complex (Ref. 7 and Fig. 4). Login to comment
216 What mechanisms could be responsible for enhanced TAP1 labeling in TAP1(K544M)/TAP2-eYFP complexes compared with TAP1(K544M)/TAP2(K509M) complexes? Login to comment
221 When we compared 8-azido-[␥-32 P]ATP binding by TAP1-eGFP in TAP1-eGFP/TAP2 complexes and TAP1-eGFP/ TAP2(K509M) complexes, we found that the TAP1-eGFP labeling intensity was enhanced when in complex with wild type TAP2 compared with TAP2(K509M). Login to comment
224 We observed that the derived affinity corresponding to TAP1(K544M) labeling in TAP1(K544M)/TAP2-eYFP complexes was nearly identical to that corresponding to TAP2-eYFP labeling (Fig. 4C) and significantly higher than that measured in TAP1(K544M)/TAP2(K509M) (Fig. 4E, middle panel; KD cannot be estimated because significant labeling was not visualized). Login to comment
225 Thus, the TAP2(K509M) mutation appears to have distinct effects on labeling of TAP1 compared with TAP1(K544M). Login to comment
237 High affinity labeling of TAP1(K544M) residues in TAP1(K544M)/TAP2-eYFP complexes (Fig. 4), but not of TAP2(K509M) residues in TAP1-eGFP/TAP2(K509M) complexes (Fig. 2), might arise because of conformational differences between the two mutant complexes. Login to comment
250 The analyses undertaken here also allow for a reassessment of the effects of TAP1(K544M) and TAP2(K509M) mutations upon peptide binding to TAP1/TAP2 complexes. Login to comment
252 Both mutant complexes were found to bind TAP-specific peptides with high affinity at room temperature (5); however, whereas the binding affinity of the TAP1(K544M)/TAP2 complex (KD ϭ 17.4 Ϯ 4.8 nM) was very similar to wild type (KD ϭ 19.4 Ϯ 4.8 nM), the affinity of the TAP1/TAP2(K509M) was ϳ2-fold reduced (KD ϭ 39.2 Ϯ 5.9 nM). Login to comment
45 EXPERIMENTAL PROCEDURES Baculoviruses for Expression of TAP1, TAP2, TAP1(K544M), TAP2(K509M), T2MT1C, T1MT2C, TAP1-eGFP, and TAP2-eYFP- Baculoviruses encoding wild type human TAP1 and TAP2 were obtained from the laboratory of Dr. Robert Tampe &#b4; (22). Login to comment
98 Mutation of the TAP2 Walker A lysine residue (TAP2(K509M)) reduced the TAP2-associated signal and derived affinity (KD b0e; 20 òe;M when expressed in complex with TAP1-eGFP) (Fig. 2, E, bottom panel compared with top panel, and H and Table I). Login to comment
134 Mutation of the TAP2 Walker A lysine TAP2(K509M) reduced TAP2 labeling and the corresponding affinity. Login to comment
136 The binding affinity corresponding to TAP1-eGFP labeling when in complex with wild type TAP2 is similar to that derived when TAP1 is in complex with the nucleotide binding-deficient TAP2(K509M). Login to comment
138 KD (ATP) n KD (ADP) n òe;M òe;M TAP2 nucleotide binding TAP2-eYFP 19.3 afe; 2.5 2 4.4 afe; 1.4 2 TAP2/TAP1-eGFP 2.7 afe; 1.0 3 0.5 afe; 0.1 2 TAP2(K509M)/TAP1-eGFP b0e;20 4 9.0 afe; 1.4 2 TAP1 nucleotide binding TAP1 4.6 afe; 1.9 3 1.4 afe; 0.1 2 TAP1-eGFP/TAP2 2.1 afe; 0.8 3 0.7 afe; 0.1 3 TAP1-eGFP/TAP2(K509M) 2.8 afe; 2.9 4 0.6 afe; 0.2 3 Nucleotide Binding by the TAP1/TAP2 Complex The data shown in Fig. 2 were derived from analyses of 8-azido-[ॹ-32 P]ATP binding to TAP1/TAP2 complexes. Login to comment
162 For these analyses, we prepared microsomes containing TAP1(K544M)/ TAP2 complexes and TAP1(K544M)/TAP2(K509M) complexes. Login to comment
164 At comparable expression levels of both components (Fig. 4D), strong labeling was visualized for the TAP1(K544M)/TAP2 combination, whereas signals for the TAP1(K544M)/TAP2(K509M) combination were barely detectable (Fig. 4E, top and middle panels, respectively). Login to comment
174 eYFP complexes (Fig. 4E, bottom panel), a corresponding distinct signal was not visualized in the TAP1(K544M)/ TAP2(K509M) combination (Fig. 4E, middle panel), despite the higher expression of TAP1(K544M) in the latter complexes. Login to comment
185 Using TAP1-eGFP/TAP2(K509M) complexes under conditions of TAP1 excess or TAP2 excess (Fig. 2), we determined that the two nucleotide-binding sites of TAP1/TAP2 complexes did in fact bind 8-azido-ATP with apparent affinities that were, within the error of these measurements, quite similar to each other. Login to comment
202 The absence of a signal was not due to the expression level, as TAP1(K544M) was expressed at higher levels in the microsomes with the TAP2(K509M) combination compared with the TAP2-eYFP combination (see D). Login to comment
210 Mutation of the TAP2 Walker A lysine residue (TAP2(K509M)) indeed influenced nucleotide binding at the TAP2 site (Fig. 2). Login to comment
215 However, co-expression of TAP1(K544M) with TAP2(K509M) resulted in a nucleotide-binding deficient complex (Ref. 7 and Fig. 4). Login to comment
217 What mechanisms could be responsible for enhanced TAP1 labeling in TAP1(K544M)/TAP2-eYFP complexes compared with TAP1(K544M)/TAP2(K509M) complexes? Login to comment
222 When we compared 8-azido-[ॹ-32 P]ATP binding by TAP1-eGFP in TAP1-eGFP/TAP2 complexes and TAP1-eGFP/ TAP2(K509M) complexes, we found that the TAP1-eGFP labeling intensity was enhanced when in complex with wild type TAP2 compared with TAP2(K509M). Login to comment
226 Thus, the TAP2(K509M) mutation appears to have distinct effects on labeling of TAP1 compared with TAP1(K544M). Login to comment
238 High affinity labeling of TAP1(K544M) residues in TAP1(K544M)/TAP2-eYFP complexes (Fig. 4), but not of TAP2(K509M) residues in TAP1-eGFP/TAP2(K509M) complexes (Fig. 2), might arise because of conformational differences between the two mutant complexes. Login to comment
251 The analyses undertaken here also allow for a reassessment of the effects of TAP1(K544M) and TAP2(K509M) mutations upon peptide binding to TAP1/TAP2 complexes. Login to comment
253 Both mutant complexes were found to bind TAP-specific peptides with high affinity at room temperature (5); however, whereas the binding affinity of the TAP1(K544M)/TAP2 complex (KD afd; 17.4 afe; 4.8 nM) was very similar to wild type (KD afd; 19.4 afe; 4.8 nM), the affinity of the TAP1/TAP2(K509M) was b03;2-fold reduced (KD afd; 39.2 afe; 5.9 nM). Login to comment

[hide] Walker A lysine mutations of TAP1 and TAP2 interfe... J Biol Chem. 2001 Mar 9;276(10):7526-33. Epub 2000 Nov 30. Lapinski PE, Neubig RR, Raghavan M
Walker A lysine mutations of TAP1 and TAP2 interfere with peptide translocation but not peptide binding.
J Biol Chem. 2001 Mar 9;276(10):7526-33. Epub 2000 Nov 30., [PMID:11099504]

Abstract [show]
We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1.TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M).TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.

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No. Sentence Comment
2 Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. Login to comment
7 Peptide translocation is undetectable for TAP1⅐TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M)⅐TAP2 complexes. Login to comment
44 Toward a definition of the requirement for nucleotide binding and hydrolysis by each TAP subunit for peptide binding and translocation, we generated mutants of TAP1 (K544M) and TAP2 (K509M) that were altered at a conserved lysine residue of the Walker A motif (GXXGXGK(S/T)) of each protein. Login to comment
47 We observe that the Walker A lysine mutations in TAP1 and TAP2 have distinct effects upon nucleotide binding to each subunit, with nucleotide binding being significantly impaired in the TAP1(K544M) mutant but not in the TAP2(K509M) mutant. Login to comment
111 Recombinant baculoviruses were generated encoding histidine-tagged TAP1 (TAP1-His), the corresponding Walker A lysine mutant (TAP1(K544M)-His), and the TAP2 Walker A lysine mutant (TAP2(K509M)). Login to comment
114 For comparisons of nucleotide binding by each mutant or wild type TAP subunit, insect cells were infected with baculoviruses encoding TAP1-His, TAP1(K544M)- His, TAP2, or TAP2(K509M) for ϳ72 h. Login to comment
121 By contrast, TAP2(K509M) binding to ATP and ADP beads does not appear to be significantly impaired relative to wild type TAP2 (Fig. 1B, lanes 1 and 2 compared with lanes 5 and 6). Login to comment
128 Likewise, TAP2(K509M) associates with TAP1 as does wild type TAP2, as measured by coimmunoprecipitation analyses with the TAP1-specific antibody 148.3 (anti-TAP1) (9) and anti-TAP2 (Fig. 2B). Login to comment
139 Binding of TAP1-His, TAP1(K544M)-His, TAP2, and TAP2(K509M) to nucleotide-agarose beads. Login to comment
143 B, lanes 1 and 2 show that TAP2 binds to ATP and ADP. Lanes 5 and 6 show that the binding pattern for TAP2(K509M) is similar to wild type TAP2 and indicate that the mutant is not deficient in nucleotide binding. Login to comment
144 (K509M) complexes were impaired for translocation. Login to comment
145 Impaired translocation by TAP1⅐TAP2(K509M) complexes was not due to reduced expression of either TAP1 or TAP2 (Fig. 3B). Login to comment
146 Indeed, microsomes containing TAP1-His150;TAP2 complexes yielded higher cpmϩATP/cpm-ATP ratios, although the expression levels of TAP1 and TAP2 were significantly lower than that present in microsomal preparations of TAP1⅐TAP2(K509M) complexes (Fig. 3B, compare lane 2 with lane 3). Login to comment
158 In the experiments shown in Fig. 4, A and C, the same microsome preparations of TAP1⅐TAP2 and TAP1⅐TAP2(K509M) were used as for the translocation assays shown in Fig. 3 (Fig. 3B, lanes 1 and 2). Login to comment
165 B, TAP1⅐TAP2, or TAP1⅐TAP2(K509M) interactions. Login to comment
172 In three independent translocation experiments, the average cpmϩATP/cpm-ATP ratios for TAP1(K544M)-His⅐TAP2 complexes were 2-fold higher than for single subunit controls, whereas the average cpmϩATP/cpm-ATP ratios for TAP1⅐TAP2(K509M) complexes were at the same level as the single subunit controls. Login to comment
175 The resulting blot shows that while no translocation signal is observable for TAP1⅐TAP2(K509M) complexes, both TAP subunits are expressed at levels comparable with the wild type complex (compare lanes 1 and 2). Login to comment
198 For wild type TAP1⅐TAP2 and TAP1⅐TAP2(K509M), the same microsomes were used as in the translocation assays indicated in Fig. 3. Login to comment
200 The total protein concentration on each microsome preparation was 0.7 mg/ml TAP1⅐TAP2 (A), 1.8 mg/ml TAP1(K544M)-His⅐TAP2 (B), 1.3 mg/ml TAP1⅐TAP2(K509M) (C), and 1.3 mg/ml uninfected (D). Login to comment
204 The calculated binding constants for the TAP1⅐TAP2(K509M) mutant indicated that the peptide binding affinity of this mutant was slightly weaker compared with wild type TAP complexes. Login to comment
215 We observed that the TAP1(K544M) mutation significantly reduced nucleotide binding by TAP1 but that the TAP2(K509M) mutation did not significantly alter nucleotide binding by TAP2. Login to comment
220 Here we report that the K509M mutation in TAP2 abrogates peptide transport by TAP1⅐TAP2(K509M) complexes, although ATP binding by this mutant is not significantly different from wild type. Login to comment
221 Impairment in peptide translocation does not arise from structural disruptions induced by the mutation, since TAP1⅐TAP2(K509M) complexes are capable of binding both peptides and nucleotides (Figs. 1 and 3). Login to comment
222 Furthermore, based upon limited proteolytic digestion analysis, the proteolysis profiles observed for TAP1⅐TAP2(K509M) complexes closely parallel the profiles seen for TAP1⅐TAP2 complexes.2 Thus, nucleotide hydrolysis by TAP complexes containing mutant TAP2 appears to be impaired. Login to comment
231 The total protein concentration on each microsome preparation was 0.7 mg/ml TAP1⅐TAP2 (A), 1.1 mg/ml TAP1(K544M)-His⅐TAP2 (B), and 1.3 mg/ml TAP1⅐TAP2(K509M) (C). Login to comment
234 By similar criteria, four separate sets of experiments comparing peptide binding by TAP1⅐TAP2(K509M) microsomes and uninfected microsomes verify that this mutant complex is also capable of binding peptides. Login to comment
244 The observation that the TAP2(K509M) mutation impairs translocation by TAP1⅐TAP2(K509M) complexes although no residue alterations were introduced into TAP1 indicates that the ATPase activity at TAP1, if present, is insufficient for completion of a peptide translocation cycle. Taken together with the observation that the TAP1(K544M) mutation significantly reduces peptide translocation efficiency of TAP complexes when no residue modifications are introduced into TAP2, these results indicate a coupling between nucleotide interactions with TAP1 and TAP2. Login to comment
259 It is interesting that the TAP2(K509M) mutation abrogates peptide translocation by TAP1⅐TAP2(K509M) complexes but that the TAP1 mutant with a significant impairment in TAP1 nucleotide binding appears to, with low efficiency, mediate peptide translocation by TAP1(K544M)-His⅐TAP2 complexes. Login to comment
268 Using similar sets of fluorescence quenching assays, we show here that TAP1(K544M)- His⅐TAP2 and TAP1⅐TAP2(K509M) complexes are capable of binding peptides, although the binding affinity of TAP1⅐TAP2(K509M) complexes appears weaker than wild type. Login to comment
112 Recombinant baculoviruses were generated encoding histidine-tagged TAP1 (TAP1-His), the corresponding Walker A lysine mutant (TAP1(K544M)-His), and the TAP2 Walker A lysine mutant (TAP2(K509M)). Login to comment
115 For comparisons of nucleotide binding by each mutant or wild type TAP subunit, insect cells were infected with baculoviruses encoding TAP1-His, TAP1(K544M)- His, TAP2, or TAP2(K509M) for b03;72 h. Login to comment
122 By contrast, TAP2(K509M) binding to ATP and ADP beads does not appear to be significantly impaired relative to wild type TAP2 (Fig. 1B, lanes 1 and 2 compared with lanes 5 and 6). Login to comment
129 Likewise, TAP2(K509M) associates with TAP1 as does wild type TAP2, as measured by coimmunoprecipitation analyses with the TAP1-specific antibody 148.3 (anti-TAP1) (9) and anti-TAP2 (Fig. 2B). Login to comment
140 Binding of TAP1-His, TAP1(K544M)-His, TAP2, and TAP2(K509M) to nucleotide-agarose beads. Login to comment
147 Indeed, microsomes containing TAP1-HisዼTAP2 complexes yielded higher cpmaf9;ATP/cpmafa;ATP ratios, although the expression levels of TAP1 and TAP2 were significantly lower than that present in microsomal preparations of TAP1ዼTAP2(K509M) complexes (Fig. 3B, compare lane 2 with lane 3). Login to comment
159 In the experiments shown in Fig. 4, A and C, the same microsome preparations of TAP1ዼTAP2 and TAP1ዼTAP2(K509M) were used as for the translocation assays shown in Fig. 3 (Fig. 3B, lanes 1 and 2). Login to comment
166 B, TAP1ዼTAP2, or TAP1ዼTAP2(K509M) interactions. Login to comment
173 In three independent translocation experiments, the average cpmaf9;ATP/cpmafa;ATP ratios for TAP1(K544M)-HisዼTAP2 complexes were 2-fold higher than for single subunit controls, whereas the average cpmaf9;ATP/cpmafa;ATP ratios for TAP1ዼTAP2(K509M) complexes were at the same level as the single subunit controls. Login to comment
176 The resulting blot shows that while no translocation signal is observable for TAP1ዼTAP2(K509M) complexes, both TAP subunits are expressed at levels comparable with the wild type complex (compare lanes 1 and 2). Login to comment
199 For wild type TAP1ዼTAP2 and TAP1ዼTAP2(K509M), the same microsomes were used as in the translocation assays indicated in Fig. 3. Login to comment
201 The total protein concentration on each microsome preparation was 0.7 mg/ml TAP1ዼTAP2 (A), 1.8 mg/ml TAP1(K544M)-HisዼTAP2 (B), 1.3 mg/ml TAP1ዼTAP2(K509M) (C), and 1.3 mg/ml uninfected (D). Login to comment
205 The calculated binding constants for the TAP1ዼTAP2(K509M) mutant indicated that the peptide binding affinity of this mutant was slightly weaker compared with wild type TAP complexes. Login to comment
216 We observed that the TAP1(K544M) mutation significantly reduced nucleotide binding by TAP1 but that the TAP2(K509M) mutation did not significantly alter nucleotide binding by TAP2. Login to comment
223 Furthermore, based upon limited proteolytic digestion analysis, the proteolysis profiles observed for TAP1ዼTAP2(K509M) complexes closely parallel the profiles seen for TAP1ዼTAP2 complexes.2 Thus, nucleotide hydrolysis by TAP complexes containing mutant TAP2 appears to be impaired. Login to comment
232 The total protein concentration on each microsome preparation was 0.7 mg/ml TAP1ዼTAP2 (A), 1.1 mg/ml TAP1(K544M)-HisዼTAP2 (B), and 1.3 mg/ml TAP1ዼTAP2(K509M) (C). Login to comment
235 By similar criteria, four separate sets of experiments comparing peptide binding by TAP1ዼTAP2(K509M) microsomes and uninfected microsomes verify that this mutant complex is also capable of binding peptides. Login to comment
245 The observation that the TAP2(K509M) mutation impairs translocation by TAP1ዼTAP2(K509M) complexes although no residue alterations were introduced into TAP1 indicates that the ATPase activity at TAP1, if present, is insufficient for completion of a peptide translocation cycle. Taken together with the observation that the TAP1(K544M) mutation significantly reduces peptide translocation efficiency of TAP complexes when no residue modifications are introduced into TAP2, these results indicate a coupling between nucleotide interactions with TAP1 and TAP2. Login to comment
260 It is interesting that the TAP2(K509M) mutation abrogates peptide translocation by TAP1ዼTAP2(K509M) complexes but that the TAP1 mutant with a significant impairment in TAP1 nucleotide binding appears to, with low efficiency, mediate peptide translocation by TAP1(K544M)-HisዼTAP2 complexes. Login to comment
269 Using similar sets of fluorescence quenching assays, we show here that TAP1(K544M)- HisዼTAP2 and TAP1ዼTAP2(K509M) complexes are capable of binding peptides, although the binding affinity of TAP1ዼTAP2(K509M) complexes appears weaker than wild type. Login to comment

[hide] Distinct functional properties of the TAP subunits... Curr Biol. 2001 Feb 20;11(4):242-51. Alberts P, Daumke O, Deverson EV, Howard JC, Knittler MR
Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle.
Curr Biol. 2001 Feb 20;11(4):242-51., [PMID:11250152]

Abstract [show]
BACKGROUND: The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated. RESULTS: Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2. CONCLUSIONS: Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.

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239 The authors showof T2 containing rat TAP1a and rat TAP2a , which have been described that mutation K607M in TAP1 and K509M in TAP2, which are differentpreviously [37], were cultured in IMDM (Gibco BRL) supplemented with from our mutations, have distinct effects on nucleotide binding and peptide 10% FCS (BIO Whittaker) and 1 mg/ml G418 (PAA, Co¨lbe). Login to comment