Pool M R, López-Huertas E, Baker A
Centre for Plant Sciences, Leeds Institute for Plant Biotechnology and Agriculture, University of Leeds, Leeds LS2 9JT, UK.
EMBO J. 1998 Dec 1;17(23):6854-62. doi: 10.1093/emboj/17.23.6854.
A hybrid protein in which the immunoglobulin G-binding domain of Staphylococcus aureus protein A replaced the N-terminal 43 amino acids of glycolate oxidase (a peroxisomal protein) was affinity purified after expression in Escherichia coli and used to study peroxisomal protein import in vitro. The fusion protein, which co-purifies with the bacterial chaperones dnaK and groEL, binds to glyoxysomes and is partially translocated in an ATP-dependent reaction which is independent of eukaryotic cytosol. Both binding and translocation are dependent upon the amount of glyoxysomes present. The partially translocated species has a transmembrane location and is extractable by salt, indicating that it is held in the membrane by ionic interactions. In the absence of ATP, the fusion protein binds to the surface of the glyoxysomes and competes the binding of authentic matrix proteins. The surface-bound protein can be chased to the transmembrane species upon the addition of ATP. These results indicate that the surface-bound form is a true translocation intermediate. The availability of this fusion protein in milligram quantities offers the possibility to use the intermediate formed in the absence of ATP and the transmembrane species to probe interactions with the peroxisome import machinery.
一种杂合蛋白，其中金黄色葡萄球菌蛋白A的免疫球蛋白G结合结构域取代了乙醇酸氧化酶（一种过氧化物酶体蛋白）的N端43个氨基酸，在大肠杆菌中表达后经亲和纯化，并用于体外研究过氧化物酶体蛋白的导入。该融合蛋白与细菌伴侣蛋白dnaK和groEL共纯化，可与乙醛酸循环体结合，并在一个依赖ATP的反应中部分转运，该反应独立于真核细胞质。结合和转运都取决于乙醛酸循环体的数量。部分转运的物种具有跨膜定位，可被盐提取，表明它通过离子相互作用固定在膜中。在没有ATP的情况下，融合蛋白与乙醛酸循环体表面结合，并竞争真实基质蛋白的结合。加入ATP后，表面结合的蛋白可被追踪到跨膜物种。这些结果表明，表面结合形式是一种真正的转运中间体。这种毫克量的融合蛋白的可得性为利用在没有ATP时形成的中间体和跨膜物种来探测与过氧化物酶体导入机制的相互作用提供了可能性。
