Lapinski P E, Neubig R R, Raghavan M
Department of Microbiology and Immunology and Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0620, USA.
J Biol Chem. 2001 Mar 9;276(10):7526-33. doi: 10.1074/jbc.M009448200. Epub 2000 Nov 30.
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.
我们构建了与抗原加工亚基TAP1和TAP2相关的转运体突变体，这些突变体在核苷酸结合域（NBD）的沃克A基序中的保守赖氨酸残基处发生了改变。在其他ATP结合盒转运体中，赖氨酸的突变已被证明会降低或消除ATP水解活性，在某些情况下还会损害核苷酸结合。突变体TAP1（K544M）和TAP2（K509M）在昆虫细胞中表达，并评估了这些突变对核苷酸结合、肽结合和肽转运的影响。相对于野生型TAP1，突变型TAP1亚基在核苷酸结合方面显著受损。相对于野生型TAP2，TAP2中的相同突变不会显著损害核苷酸结合。使用荧光猝灭测定法测量荧光肽的结合，我们发现这两个突变体与它们的野生型伴侣结合时都能结合肽。由于突变型TAP1在核苷酸结合方面显著受损，这些结果表明核苷酸与TAP1的结合不是肽与TAP复合物结合的必要条件。对于TAP1.TAP2（K509M）复合物，无法检测到肽转运，但对于TAP1（K544M）.TAP2复合物，可以检测到低水平的转运。这些结果表明，含有任一突变型TAP亚基的TAP复合物在核苷酸水解方面存在缺陷，并表明一个完整的TAP NBD不足以有效催化肽转运。综上所述，这些结果还表明在单个转运循环中TAP1和TAP2 NBD可能具有不同的功能。
