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首页> 外文期刊>Molecular cell >Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.
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Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.

机译：映射蛋白质-蛋白质相互作用的酵母ABC转运蛋白Ycf1p通过整合分裂遍在蛋白膜酵母两杂交分析。

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摘要

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 Deltaycf1 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.
机译：ATP结合盒（ABC）转运蛋白在人类健康和疾病中很重要，代表了最大的跨膜蛋白家族。然而，它们的高度疏水性使使用标准生化方法鉴定相互作用蛋白变得复杂。在这里，我们报道了使用基因组整合的“诱饵”构建体，因此命名为iMYTH的改良型分裂泛素膜酵母双杂交（MYTH）技术的开发。我们以文库筛选的形式使用了iMYTH，并确定了酵母ABC转运蛋白Ycf1p的六个潜在的相互作用伴侣。这些基因中的几个缺失的菌株导致亚砷酸盐敏感性类似于Deltaycf1菌株。转运分析表明，其中一个Tus1p是小GTPase Rho1p的鸟嘌呤核苷酸交换因子（GEF），是Ycf1p的Rho1p依赖性阳性调节子。我们的研究提供了原理性证明，即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因子和调节剂的理想方法。

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