ABCMdb

PMID: 12501238

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., [PubMed]

Sentences

No. Mutations Sentence Comment

45 ABCB3 p.Lys509Met 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 46 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met We have previously described the construction of baculoviruses encoding the TAP1 mutant (TAP1(K544M) and the TAP2 mutant (TAP2(K509M)) (5). Login to comment 98 ABCB3 p.Lys509Met 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 99 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met Mutation of the TAP2 Walker A lysine TAP2(K509M) reduced TAP2 labeling and the corresponding affinity. Login to comment 134 ABCB3 p.Lys509Met Mutation of the TAP2 Walker A lysine TAP2(K509M) reduced TAP2 labeling and the corresponding affinity. Login to comment 135 ABCB3 p.Lys509Met 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 136 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 138 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 161 ABCB3 p.Lys509Met For these analyses, we prepared microsomes containing TAP1(K544M)/ TAP2 complexes and TAP1(K544M)/TAP2(K509M) complexes. Login to comment 162 ABCB3 p.Lys509Met For these analyses, we prepared microsomes containing TAP1(K544M)/ TAP2 complexes and TAP1(K544M)/TAP2(K509M) complexes. Login to comment 163 ABCB3 p.Lys509Met 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 164 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 174 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 185 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 202 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met Mutation of the TAP2 Walker A lysine residue (TAP2(K509M)) indeed influenced nucleotide binding at the TAP2 site (Fig. 2). Login to comment 210 ABCB3 p.Lys509Met Mutation of the TAP2 Walker A lysine residue (TAP2(K509M)) indeed influenced nucleotide binding at the TAP2 site (Fig. 2). Login to comment 214 ABCB3 p.Lys509Met However, co-expression of TAP1(K544M) with TAP2(K509M) resulted in a nucleotide-binding deficient complex (Ref. 7 and Fig. 4). Login to comment 215 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 217 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 222 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 224 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met ABCB3 p.Lys509Met Thus, the TAP2(K509M) mutation appears to have distinct effects on labeling of TAP1 compared with TAP1(K544M). Login to comment 226 ABCB3 p.Lys509Met Thus, the TAP2(K509M) mutation appears to have distinct effects on labeling of TAP1 compared with TAP1(K544M). Login to comment 237 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 238 ABCB3 p.Lys509Met ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 251 ABCB3 p.Lys509Met 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 ABCB3 p.Lys509Met 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 253 ABCB3 p.Lys509Met 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