ABCMdb

PMID: 11099504

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., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met Peptide translocation is undetectable for TAP1⅐TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M)⅐TAP2 complexes. Login to comment 44 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 112 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 115 ABCB3 p.Lys509Met 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 121 ABCB3 p.Lys509Met 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 122 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 129 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met Binding of TAP1-His, TAP1(K544M)-His, TAP2, and TAP2(K509M) to nucleotide-agarose beads. Login to comment 140 ABCB3 p.Lys509Met Binding of TAP1-His, TAP1(K544M)-His, TAP2, and TAP2(K509M) to nucleotide-agarose beads. Login to comment 143 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met (K509M) complexes were impaired for translocation. Login to comment 145 ABCB3 p.Lys509Met ABCB3 p.Lys509Met Impaired translocation by TAP1⅐TAP2(K509M) complexes was not due to reduced expression of either TAP1 or TAP2 (Fig. 3B). Login to comment 146 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 147 ABCB3 p.Lys509Met 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 158 ABCB3 p.Lys509Met 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 159 ABCB3 p.Lys509Met 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 165 ABCB3 p.Lys509Met B, TAP1⅐TAP2, or TAP1⅐TAP2(K509M) interactions. Login to comment 166 ABCB3 p.Lys509Met B, TAP1ዼTAP2, or TAP1ዼTAP2(K509M) interactions. Login to comment 172 ABCB3 p.Lys509Met 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 173 ABCB3 p.Lys509Met 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 175 ABCB3 p.Lys509Met 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 176 ABCB3 p.Lys509Met 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 198 ABCB3 p.Lys509Met For wild type TAP1⅐TAP2 and TAP1⅐TAP2(K509M), the same microsomes were used as in the translocation assays indicated in Fig. 3. Login to comment 199 ABCB3 p.Lys509Met For wild type TAP1ዼTAP2 and TAP1ዼTAP2(K509M), the same microsomes were used as in the translocation assays indicated in Fig. 3. Login to comment 200 ABCB3 p.Lys509Met 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 201 ABCB3 p.Lys509Met 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 204 ABCB3 p.Lys509Met 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 205 ABCB3 p.Lys509Met 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 215 ABCB3 p.Lys509Met 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 216 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 223 ABCB3 p.Lys509Met 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 231 ABCB3 p.Lys509Met 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 232 ABCB3 p.Lys509Met 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 234 ABCB3 p.Lys509Met 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 235 ABCB3 p.Lys509Met 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 244 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 245 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 259 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 260 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 268 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 269 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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