Tomáš Petrík, Zuzana Brzáčová, Regina Sepšiová, Katarína Veljačiková, Ľubomír Tomáška
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引用次数: 0
摘要
为了评估酵母细胞对端粒缺陷的即时反应,我们使用了助剂诱导脱落子(AID),它能快速消耗必需的(Rap1、Tbf1、Cdc13、Stn1)和非必需的(Est1、Est2、Est3)端粒蛋白。通过使用两种变体的 AID 系统,我们发现所研究的大多数蛋白质都会在添加辅酶后 10-30 分钟内耗尽。正如预期的那样,如果菌株是在适当的碳源和低于 28°C 的温度下培养的,那么耗尽必需蛋白就会产生不分裂的细胞。Cdc13 和 Stn1 消耗殆尽的细胞早在添加辅酶 3 小时后就表现出单链悬垂的延伸。值得注意的是,将携带 AID 标记的必需蛋白的菌株在有辅助素存在的情况下长时间培养,会导致出现抗辅助素的克隆,至少部分原因是 OsTIR1 基因发生了突变。在评估携带 AID 标记的非必要端粒蛋白的菌株的端粒长度时,我们发现 Est1 和 Est3 的缺失会导致依赖于辅助素的端粒缩短。然而,EST3-AID菌株即使在没有辅酶的情况下端粒也略短。此外,一株带有AID标记的Est2(端粒酶的催化亚基)的菌株不仅在没有叶绿素的情况下端粒较短,而且也没有表现出叶绿素依赖性端粒缩短。我们的研究结果表明,虽然 AID 可用于评估细胞对端粒去保护的即时反应,但必须仔细评估 AID 标记对相关蛋白质特性的影响。
Pros and cons of auxin-inducible degron as a tool for regulated depletion of telomeric proteins from Saccharomyces cerevisiae.
To assess the immediate responses of the yeast cells to telomere defects, we employed the auxin-inducible degron (AID) enabling rapid depletion of essential (Rap1, Tbf1, Cdc13, Stn1) and non-essential (Est1, Est2, Est3) telomeric proteins. Using two variants of AID systems, we show that most of the studied proteins are depleted within 10-30 min after the addition of auxin. As expected, depletion of essential proteins yields nondividing cells, provided that the strains are cultivated in an appropriate carbon source and at temperatures lower than 28°C. Cells with depleted Cdc13 and Stn1 exhibit extension of the single-stranded overhang as early as 3 h after addition of auxin. Notably, prolonged incubation of strains carrying AID-tagged essential proteins in the presence of auxin resulted in the appearance of auxin-resistant clones, caused at least in part by mutations within the OsTIR1 gene. Upon assessing the length of telomeres in strains carrying AID-tagged non-essential telomeric proteins, we found that the depletion of Est1 and Est3 leads to auxin-dependent telomere shortening. However, the EST3-AID strain had slightly shorter telomeres even in the absence of auxin. Furthermore, a strain with the AID-tagged version of Est2 (catalytic subunit of telomerase) not only had shorter telomeres in the absence of auxin but also did not exhibit auxin-dependent telomere shortening. Our results demonstrate that while AID can be useful in assessing immediate cellular responses to telomere deprotection, each strain must be carefully evaluated for the effect of AID-tag on the properties of the protein of interest.
期刊介绍:
Yeast publishes original articles and reviews on the most significant developments of research with unicellular fungi, including innovative methods of broad applicability. It is essential reading for those wishing to keep up to date with this rapidly moving field of yeast biology.
Topics covered include: biochemistry and molecular biology; biodiversity and taxonomy; biotechnology; cell and developmental biology; ecology and evolution; genetics and genomics; metabolism and physiology; pathobiology; synthetic and systems biology; tools and resources