Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Mol Biol Cell. 2012 Nov;23(21):4203-11. doi: 10.1091/mbc.E12-07-0548. Epub 2012 Aug 29.
The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle-dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1-to-S phase progression. Deletion of DIA2 rescues a delay in the G1-to-S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1-to-S phase progression.
泛素蛋白酶体系统在控制细胞周期中起着关键作用。芽殖酵母 F -box 蛋白 Dia2 是基因组稳定性所必需的，并且以细胞周期依赖性方式被靶向进行泛素依赖性降解，但泛素化途径的身份尚不清楚。我们证明 Hect 结构域 E3 泛素连接酶 Tom1 是 Dia2 蛋白降解所必需的。DIA2 的缺失部分抑制了 tom1 突变体的温度敏感表型。Tom1 在细胞周期的 G1 和 G2/M 期对 Dia2 的泛素化和降解是必需的，而在 S 期 Dia2 蛋白稳定。我们发现 Tom1 与 Dia2 的结合在 G1 期增强，在 S 期减少，这表明了这种蛋白水解开关的机制。Tom1 在 Dia2 的降解/NLS 结构域中识别特定的正电荷残基。这些残基的缺失会阻止 Tom1 介导的 Dia2 周转，并导致 G1 到 S 期进程的延迟。缺失 DIA2 可挽救 tom1Δ 突变体中 G1 到 S 期转变的延迟。我们的研究结果表明，Tom1 通过识别 Dia2 降解/NLS 结构域中的正电荷残基，在细胞周期中靶向 Dia2 进行降解，并且 Dia2 蛋白降解有助于 G1 到 S 期的进展。
