Dominant negative mutations in yeast Hsp90 reveal triage decision mechanism targeting client proteins for degradation.
Dominant negative mutations in yeast Hsp90 reveal triage decision mechanism targeting client proteins for degradation.
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酵母 Hsp90 的显性负突变揭示了针对客户蛋白降解的分类决策机制。
DOI：
10.1101/2024.01.02.573950
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Bolon,DanielNA
中科院分区：
文献类型：
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作者：
Flynn,JuliaM;Joyce,MargotE;Bolon,DanielNA
细胞的大多数基本过程都是由蛋白质介导的。然而，大多数蛋白质的生物学相关机制尚不清楚。显性负突变为研究蛋白质机制提供了有价值的工具，但由于其毒性作用而难以分离。我们使用突变扫描方法来识别酵母 Hsp90 的显性失活突变。 Hsp90 是一种分子伴侣，可与许多共分子伴侣和客户蛋白形成动态复合物。体外分析阐明了 Hsp90、共伴侣和客户的一些关键生化状态和结构；然而，Hsp90的生物学机制仍不清楚。例如，高通量研究发现许多 E3 泛素连接酶与 Hsp90 结合，但尚不清楚这些酶是否主要是客户端或用于标记其他客户端进行降解。我们将 Hsp90 ATP 酶结构域中所有点突变的文库引入酵母中，并注意到在我们可以分析的最早点上，有 176 个点突变减少了 10 倍以上。耗尽突变有两个热点区域，位于 ATP 闭合环的铰链处。我们使用诱导型启动子驱动的突变库量化了铰链区突变的显性负生长效应。我们详细分析了个体显性失活突变，发现添加阻止 Hsp90 水解 ATP 的 E33A 突变消除了显性失活表型。下拉实验没有发现任何稳定的结合伴侣，表明主要效应是由动态复合物介导的。 DN Hsp90 降低了两种 Hsp90 模型客户、糖皮质激素受体 (GR) 和 v-src 激酶的表达水平。使用 MG132，我们发现 GR 以蛋白酶体依赖性方式迅速不稳定。这些发现证明 E3 连接酶与 Hsp90 的结合可能对真核生物具有基本的质量控制功能。
Most of the fundamental processes of cells are mediated by proteins. However, the biologically-relevant mechanism of most proteins are poorly understood. Dominant negative mutations have provided a valuable tool for investigating protein mechanisms but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative mutations in yeast Hsp90. Hsp90 is a chaperone that forms dynamic complexes with many co-chaperones and client proteins. In vitro analyses have elucidated some key biochemical states and structures of Hsp90, co-chaperones, and clients; however, the biological mechanism of Hsp90 remains unclear. For example, high throughput studies have found that many E3 ubiquitin ligases bind to Hsp90, but it is unclear if these are primarily clients or acting to tag other clients for degradation. We introduced a library of all point mutations in the ATPase domain of Hsp90 into yeast and noticed that 176 were more than 10-fold depleted at the earliest point that we could analyze. There were two hot spot regions of the depleted mutations that were located at the hinges of a loop that closes over ATP. We quantified the dominant negative growth effects of mutations in the hinge regions using a library of mutations driven by an inducible promoter. We analyzed individual dominant negative mutations in detail and found that addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. Pull-down experiments did not reveal any stable binding partners, indicating that the dominant effects were mediated by dynamic complexes. DN Hsp90 decreased the expression level of two model Hsp90 clients, glucocorticoid receptor (GR) and v-src kinase. Using MG132, we found that GR was rapidly destabilized in a proteasome-dependent fashion. These findings provide evidence that the binding of E3 ligases to Hsp90 may serve a quality control function fundamental to eukaryotes.