Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis
Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis
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DOI：
10.1016/j.molcel.2007.03.011
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发表时间：
2007-04-13
期刊：
影响因子：
16
通讯作者：
Stagljar, Igor
中科院分区：
文献类型：
--
作者：
Paumi, Christian M.;Menendez, Javier;Stagljar, Igor
ATP结合盒（ABC）转运蛋白在人类健康和疾病中是重要的，并且代表了最大的跨膜蛋白家族;然而，它们的高度疏水性使得使用标准生化方法来鉴定相互作用蛋白变得复杂。在这里，我们报告了一个修改版本的分裂泛素膜酵母双杂交（MYTH）技术的发展，使用基因组整合的“诱饵”结构，因此命名为iMYTH。我们使用iMYTH在图书馆筛选格式，并确定了6个潜在的相互作用的合作伙伴的酵母ABC转运蛋白Ycf 1 p。删除这些基因中的几个的菌株导致类似于Delta ycf 1菌株的亚砷酸盐敏感性。转运试验表明，其中之一，Tus 1 p，鸟嘌呤核苷酸交换因子（GEF）的小GTdR Rho 1 p，是Rho 1 p依赖的正调节Ycf 1 p。我们的研究提供了原则证据，证明iMYTH是鉴定ABC转运蛋白和其他酵母跨膜蛋白的生理相互作用物和调节剂的理想方法。
The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p. Strains deleted for several of these genes result in arsenite sensitivity similar to a Delta ycf1 strain. Transport assays show that one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for the small GTPase Rho1p, is a Rho1p-dependent- positive regulator of Ycf1p. Our study provides proof of principle that iMYTH is an ideal methodology to identify physiological interactors and regulators of ABC transporters and other yeast transmembrane proteins.