A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD.
A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD.
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DOI：
10.1007/s00294-022-01227-1
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发表时间：
2022-04
期刊：
影响因子：
2.5
通讯作者：
中科院分区：
文献类型：
--
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内质网(ER)中的错误折叠蛋白被逆转录到胞浆中，泛素化并被蛋白酶体降解。在这个被称为内质网相关降解(ERAD)的过程中，内质网包埋的Hrd1泛素连接酶在识别、泛化和重新定位大量的腔蛋白和整膜蛋白方面发挥了核心作用。为了更好地确定酿酒酵母中Hrd1功能的潜在机制，已经开发了几种模型底物。其中一个底物是Sec61-2，它是Sec61易位通道的温度敏感等位基因。表达Sec61-2的细胞在25℃下生长，因为蛋白质是稳定的，但Sec61-2酵母在38℃下不能存活，因为突变的蛋白质以依赖于Hrd1的方式降解。因此，删除Hrd1稳定了Sec61-2，因此Sec61-2hrd1双突变体在38℃下是可行的。这一独特的表型使我们能够对Hrd1中的功能缺失等位基因进行无偏见的筛选。基于其在介导底物逆转录易位中的重要性，该筛查也被开发成关注编码hrd1‘S跨膜富含结构域的序列突变。最终，在Hrd1中发现了一组隐性突变，包括导致Hrd1传递到ERAD途径的一系列不稳定突变。一个更稳定的突变体存在于一个隐藏的跨膜区，但在表达该突变体的酵母中，Hrd1复合体被破坏。综上所述，这些数据证实了Hrd1复合体在ERAD过程中完整性的重要性，表明跨膜结构域之间的变构相互作用调节Hrd1复合体的形成，并为该领域提供了新的工具来定义底物逆转移位过程中ERAD组分之间的动态相互作用。
Misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for ubiquitination and degradation by the proteasome. During this process, known as ER-associated degradation (ERAD), the ER-embedded Hrd1 ubiquitin ligase plays a central role in recognizing, ubiquitinating, and retrotranslocating scores of lumenal and integral membrane proteins. To better define the mechanisms underlying Hrd1 function in Saccharomyces cerevisiae, several model substrates have been developed. One substrate is Sec61–2, a temperature sensitive allele of the Sec61 translocation channel. Cells expressing Sec61–2 grow at 25 °C because the protein is stable, but sec61–2 yeast are inviable at 38 °C because the mutated protein is degraded in a Hrd1-dependent manner. Therefore, deleting HRD1 stabilizes Sec61–2 and hence sec61–2hrd1△ double mutants are viable at 38 °C. This unique phenotype allowed us to perform a non-biased screen for loss-of-function alleles in HRD1. Based on its importance in mediating substrate retrotranslocation, the screen was also developed to focus on mutations in sequences encoding Hrd1’s transmembrane-rich domain. Ultimately, a group of recessive mutations was identified in HRD1, including an ensemble of destabilizing mutations that resulted in the delivery of Hrd1 to the ERAD pathway. A more stable mutant resided in a buried transmembrane domain, yet the Hrd1 complex was disrupted in yeast expressing this mutant. Together, these data confirm the importance of Hrd1 complex integrity during ERAD, suggest that allosteric interactions between transmembrane domains regulate Hrd1 complex formation, and provide the field with new tools to define the dynamic interactions between ERAD components during substrate retrotranslocation.
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影响因子：
33.6
作者：
Guerriero CJ;Brodsky JL
通讯作者：
Brodsky JL
DOI：
10.1073/pnas.0812114106
发表时间：
2009-06-23
影响因子：
11.1
作者：
Adle, David J.;Wei, Wenzhong;Lee, Jaekwon
通讯作者：
Lee, Jaekwon
影响因子：
2.6
作者：
Hentges, P;Van Driessche, B;Carr, AM
通讯作者：
Carr, AM
影响因子：
11.4
作者：
Flury, I;Garza, R;Hampton, RY
通讯作者：
Hampton, RY
DOI：
10.15252/embj.2020104863
发表时间：
2020-11-16
期刊：
The EMBO journal
影响因子：
--
作者：
Lips C;Ritterhoff T;Weber A;Janowska MK;Mustroph M;Sommer T;Klevit RE
通讯作者：
Klevit RE