Analysis of a conditional degradation signal in yeast and mammalian cells
Analysis of a conditional degradation signal in yeast and mammalian cells
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DOI：
10.1046/j.1432-1327.1999.00024.x
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发表时间：
1999-01-01
期刊：
影响因子：
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通讯作者：
Varshavsky, A
中科院分区：
文献类型：
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作者：
Lévy, F;Johnston, JA;Varshavsky, A
N 端规则途径是一种泛素依赖性蛋白水解系统，其目标包括带有不稳定 N 端残基的蛋白质。后者是退化信号的一部分，称为 N-degron。 Arg-DHFRts 是一种工程化的 N 端规则底物，带有 N 端精氨酸（一种不稳定残基）和 DHFRts [温度敏感的小鼠二氢叶酸还原酶 (DHFR) 部分]。先前的研究表明，在酿酒酵母中，Arg-DHFRts 在 23 摄氏度下寿命较长，但在 37 摄氏度下寿命较短。在目前的工作中，我们扩展了这一分析，发现Arg-DHFRts的降解可以在体内被甲氨蝶呤（MTX）（DHFR的低M-r配体）几乎完全抑制。在酿酒酵母中。 Arg-DHFRts 在 37 摄氏度下仅通过 N 端规则途径降解，而在小鼠细胞中，相同温度下的相同蛋白质也通过构象扰动的 DHFRts 部分中的降解决定子被另一个蛋白水解系统靶向。在小鼠细胞中，MTX 通过 DHFRts 部分内的降解决定子完全抑制 Arg-DHFRts 的降解，但仅通过 N-降解决定子部分抑制降解。当 Arg-DHFRts 的 N 末端用缺乏赖氨酸的 42 个残基延伸时，称为 e(Delta K)，所得 Arg-e(Delta K)-DHFRts 在 23 摄氏度和 37 摄氏度下快速降解。此外，与 Arg-DHFRts 相比，Arg-e(Delta K) DHFRts 的降解不能被MTX，表明 Arg-DHFRts 在 23°C 时的代谢稳定性至少部分是由于其 N 末端精氨酸的空间不可接近性造成的。 Arg-DHFRts 的 N-降解决定子是便携式降解信号的第一个例子，其活性可以通过细胞穿透化合物在体内调节。我们讨论了这一进展的影响以及泛素系统的靶向机制。
The N-end rule pathway is a ubiquitin-dependent proteolytic system, the targets of which include proteins that bear destabilizing N-terminal residues. The latter are a part of the degradation signal called the N-degron. Arg-DHFRts, an engineered N-end rule substrate, bears N-terminal arginine (a destabilizing residue) and DHFRts [a temperature-sensitive mouse dihydrofolate reductase (DHFR) moiety]. Previous work has shown that Arg-DHFRts is long-lived at 23 degrees C but short-lived at 37 degrees C in the yeast Saccharomyces cerevisiae. In the present work, we extended this analysis, and found that the degradation of Arg-DHFRts can be nearly completely inhibited in vivo by methotrexate (MTX), a low-M-r ligand of DHFR. In S. cerevisiae. Arg-DHFRts is degraded at 37 degrees C exclusively by the N-end rule pathway, whereas in mouse cells the same protein at the same temperature is also targeted by another proteolytic system, through a degron in the conformationally perturbed DHFRts moiety. In mouse cells, MTX completely inhibits the degradation of Arg-DHFRts through its degron within the DHFRts moiety, but only partially inhibits degradation through the N-degron. When the N-terminus of Arg-DHFRts was extended with a 42-residue lysine-lacking extension, termed e(Delta K), the resulting Arg-e(Delta K)-DHFRts was rapidly degraded at both 23 degrees C and 37 degrees C. Moreover, the degradation of Arg-e(Delta K) DHFRts, in contrast with that of Arg-DHFRts, could not be inhibited by MTX, suggesting that the metabolic stability of Arg-DHFRts at 23 degrees C results, at least in part, from steric inaccessibility of its N-terminal arginine. The N-degron of Arg-DHFRts is the first example of a portable degradation signal the activity of which can be modulated in vivo by a cell-penetrating compound. We discuss implications of this advance and the mechanics of targeting by the ubiquitin system.