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首页> 美国卫生研究院文献>Eukaryotic Cell >Mapping of an Internal Protease Cleavage Site in the Ssy5p Component of the Amino Acid Sensor of Saccharomyces cerevisiae and Functional Characterization of the Resulting Pro- and Protease Domains by Gain-of-Function Genetics
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Mapping of an Internal Protease Cleavage Site in the Ssy5p Component of the Amino Acid Sensor of Saccharomyces cerevisiae and Functional Characterization of the Resulting Pro- and Protease Domains by Gain-of-Function Genetics

机译：酿酒酵母氨基酸传感器的Ssy5p组件中的内部蛋白酶切割位点的映射和功能增益遗传学对所得蛋白酶和蛋白酶域的功能表征

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摘要

Ssy5p is a 77-kDa protein believed to be a component of the SPS amino acid sensor complex in the plasma membrane of Saccharomyces cerevisiae. Ssy5p has been suggested to be a chymotrypsin-like serine protease that activates the transcription factor Stp1p upon exposure of the yeast to extracellular amino acid. Here we overexpressed and partially purified Ssy5p to improve our understanding of its structure and function. Antibodies against Ssy5p expressed in Escherichia coli were isolated and used to detect Ssy5p processing in S. cerevisiae cells. Partial purification and N-terminal sequencing of processed Ssy5p revealed in vivo cleavage of Ssy5p between amino acids 381 and 382. We also isolated constitutively signaling SSY5 mutants and quantified target promoter activation and Stp1p processing. One mutant contained an amino acid substitution in the prodomain, whereas three others harbored amino acid substitutions in the protease domain. Dose-response analysis indicated that all four mutants exhibited increased basal levels of Stp1p processing. Interestingly, whereas the three constitutive mutants mapping to the protease domain of Ssy5p exhibited the decreased 50% effective concentration (EC50) characteristic of constitutive mutations previously found in Ssy1p, Ptr3p, and Ssy5p, the EC50 of the mutation that maps to the prodomain of Ssy5p remained essentially unchanged. In a model of Ssy5p derived from its similarities with α-lytic protease from Lysobacter enzymogenes, the sites corresponding to the mutations in the protease domain are clustered in a region facing the prodomain, suggesting that this region interacts with the prodomain and participates in the conformational dynamics of sensing.
机译：Ssy5p是一种77 kDa的蛋白质，被认为是酿酒酵母质膜中SPS氨基酸传感器复合物的组成部分。 Ssy5p被认为是一种胰凝乳蛋白酶样丝氨酸蛋白酶，在酵母暴露于细胞外氨基酸后会激活转录因子Stp1p。在这里，我们过表达并部分纯化了Ssy5p，以增进我们对其结构和功能的了解。分离了针对在大肠杆菌中表达的Ssy5p的抗体，并将其用于检测酿酒酵母细胞中的Ssy5p加工。部分加工的Ssy5p的纯化和N端测序揭示了Ssy5p在381和382位氨基酸之间的体内裂解。我们还分离出SSY5组成型信号转导子，并定量了靶启动子的激活和Stp1p的加工。一个突变体在前结构域中包含氨基酸取代，而其他三个突变体在蛋白酶结构域中具有氨基酸取代。剂量反应分析表明，所有四个突变体均显示出增加的Stp1p加工基础水平。有趣的是，虽然映射到Ssy5p蛋白酶结构域的三个组成性突变体表现出先前在Ssy1p，Ptr3p和Ssy5p中发现的组成性突变的50％有效浓度（EC50）特征降低，但映射到Ssy5p的结构域的突变的EC50基本上保持不变。在一个Ssy5p模型中，其与溶菌酶基因的α-分解蛋白酶相似，在蛋白酶结构域中与突变对应的位点聚集在面向前结构域的区域中，表明该区域与前结构域相互作用并参与构象。感测动力学。

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