Rapid Depletion of Budding Yeast Proteins by Fusion to a Heat-Inducible Degron
Rapid Depletion of Budding Yeast Proteins by Fusion to a Heat-Inducible Degron
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DOI：
10.1126/stke.2232004pl8
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发表时间：
2004-03
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影响因子：
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通讯作者：
Alberto Sanchez-Diaz;Masato T. Kanemaki;Vanessa Marchesi;K. Labib
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作者：
Alberto Sanchez-Diaz;Masato T. Kanemaki;Vanessa Marchesi;K. Labib
研究体内蛋白质生物学功能的一种有效方法是将其固定下来，看看细胞发生了什么。对于在细胞活力方面被抑制的蛋白质，可以简单地从其染色体基因座中删除相应的基因。然而，对必需蛋白质的研究更具挑战性，因为蛋白质的功能必须有条件地失活。在这里，我们描述了一种方法，允许靶蛋白被快速和有条件地耗尽，以便可以检查对细胞的直接影响。芽殖酵母基因的染色体位点被修饰，使得“热诱导降解决定子盒”被添加到编码蛋白的N末端，当细胞从24° C转移到37°C时，使其通过特定的泛素介导的途径降解。降解需要通过进化上保守的Ubr1蛋白识别降解决定子盒，Ubr1蛋白与泛素缀合酶相关。为了促进靶蛋白的快速和有条件的消耗，我们使用酵母菌株，其中UBR 1基因的表达可以被抑制或强烈诱导。降解决定子菌株通过使用聚合酶链反应的简单“一步”方法构建。
One effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell. For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus. The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally. Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined. The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24° to 37°C. Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme. To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the UBR1 gene can be either repressed or strongly induced. Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.