Walker A lysine mutations of TAP1 and TAP2 interfere with peptide translocation but not peptide binding.
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.
Lapinski
P E
PE
Department of Microbiology and Immunology and Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0620, USA.
Neubig
R R
RR
Raghavan
M
M
D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D000244:Adenosine Diphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D001081:Apyrase / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D021381:Protein Transport; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D010455:Peptides; C071692:TAP2 protein, human; D000244:Adenosine Diphosphate; D001081:Apyrase; D008239:Lysine