利用蛋白标签纯化酿酒酵母RAVE V1复合物

摘要

The regulator of the proton-translocating ATPases of the Vacuolar and Endosomal membranes ( RAVE) ,is an essential factor for the reversible assembly of vacuolar proton-translocating ATPases. In yeast cells, when glucose was exhausted, the V-ATPase decomposited into V1, V0, and the RAVE combined with V1 to form the complex in the cytoplasm. By using the affinity chromatography, RAVE-V1 was purified through adding the FLAG tags to the C terminal of Rav2p, which was useful for electron microscopy to study the three-dimensional structure of RAVE-V1 complex. The results showed that adding FLAG tag to the C end of Rav2p could purify RAVE-V1 complex. In addition, the interaction relationship between Leu1p with RAVE was discovered through mass spectrometry,which made the RAVE research to a new direction.%RAVE（ regulator of the H＋-ATPase of the vacuolar and endosomal membranes）是调节液泡ATP酶（ V ATP酶）装配与拆卸过程的调节酶，由Rav1p、Rav2p和Skp1p 3个亚基构成。在酿酒酵母细胞中，当葡萄糖耗尽时，V ATP酶分解成V1、V0两部分，此时，RAVE与V1以复合物的形式存在于细胞质中。本研究利用同源重组技术，构建在基因RAV2的3′端定点插入FLAG标签的重组菌株BY4742 RAV2 FLAG，通过亲和层析原理纯化RAVE V1复合物，为后续利用电子显微镜对其进行三维结构研究奠定坚实的基础。结果表明：FLAG标签添加到Rav2p的C端可以成功纯化出RAVE V1复合物；结合质谱鉴定首次发现了Leu1p与RAVE存在相互作用关系，这使得对RAVE的研究转向一个全新的方向；此外，本研究方案对其他调节蛋白及与之相互作用的蛋白组的分离纯化具有借鉴意义。