Using the cre-lox recombination system to assess functional impairment caused by amino acid substitutions in yeast proteins.
Using the cre-lox recombination system to assess functional impairment caused by amino acid substitutions in yeast proteins.
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DOI：
10.1251/bpo91
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发表时间：
2004
影响因子：
6.4
通讯作者：
Culbertson MR
中科院分区：
文献类型：
--
作者：
Shirley RL;Richards MR;Culbertson MR
开发了一种方法来评估酵母Upf3p中序列基序的功能意义，Upf3p是无义介导的mRNA衰变（NMD）所需的蛋白质。该基序位于Upf3p-Upf2p相互作用结构域的边缘，但同时类似于在结合Crm1p输出蛋白的蛋白质中发现的典型的富含亮氨酸的核输出序列（内斯）。为了测试推定的内斯的功能，首先选择引起保守NES-A残基取代的定点突变来鉴定超形态等位基因。接下来，使用细菌cre重组酶识别的loxP位点，将来自在酵母中起作用的HIV-1 Rev蛋白的可携带的Crm1 p-结合内斯ES与变体Upf 3蛋白的C-末端进行内切融合。最后，选择在NMD中有功能的变体Upf3-Rev蛋白，并检查NES-A中存在的氨基酸取代类型。突变分析表明，Upf3内斯的氨基酸取代损害核输出和Upf2 p-Upf3 p的相互作用，这两者都是Upf3 p在NMD中发挥功能所必需的。本报告中描述的方法可以修改为各种便携式蛋白质结构域的遗传分析。
A method was developed to assess the functional significance of a sequence motif in yeast Upf3p, a protein required for nonsense-mediated mRNA decay (NMD). The motif lies at the edge of the Upf3p-Upf2p interaction domain, but at the same time resembles the canonical leucine-rich nuclear export sequence (NES) found in proteins that bind Crm1p exportin. To test the function of the putative NES, site-directed mutations that cause substitutions of conserved NES-A residues were first selected to identify hypermorphic alleles. Next, a portable Crm1p-binding NES from HIV-1 Rev protein that functions in yeast was fused en masse to the C-terminus of variant Upf3 proteins using loxP sites recognized by bacterial cre-recombinase. Finally, variant Upf3-Rev proteins that were functional in NMD were selected and examined for the types of amino acid substitutions present in NES-A. The mutational analysis revealed that amino acid substitutions in the Upf3 NES impair both nuclear export and the Upf2p-Upf3p interaction, both of which are required for Upf3p to function in NMD. The method described in this report could be modified for the genetic analysis of a variety of portable protein domains.