Department of Molecular Biology, Defence Institute of Physiology and Allied Sciences, Timarpur, Delhi, 110054, India.
Yeast. 2011 Nov;28(11):783-93. doi: 10.1002/yea.1904. Epub 2011 Oct 14.
Ubiquitin conjugating enzyme 1 (Ubc1) is a member of the E2 family of enzymes that conjugates ubiquitin to damaged proteins destined for degradation by the ubiquitin proteasomal system. It is necessary for stress tolerance and is essential for cell survival in Saccharomyces cerevisiae. Ubc1 has five serine residues that are potential substrates for phosphorylation by kinases. However, no data are available to indicate that Ubc1 function or stress tolerance in S. cerevisiae is regulated by serine phosphorylation of Ubc1. We demonstrate that Ubc1 is phosphorylated in serine residue(s). Furthermore, expression of Ubc1 mutants that are 'constitutively phosphorylated' or 'dephosphorylated' in mitogen-activated protein (MAP) kinase serine residues (S97 and S115) affected tolerance to thermal and reductive stress in S. cerevisiae. Specifically, expression of Ubc1S97A and S115D increased thermo-tolerance in both BY4741 and TetO7 -UBC1ura3Δ cells. Serine phosphorylation of Ubc1 was decreased in BY4741 cells following exposure at 40 °C. Tolerance to reductive stress in the same strains correlated with the expression of Ubc1S97A. Ubc1 phosphorylation did not show significant alteration under similar conditions. Both hog1Δ and slt2Δ cells expressing Ubc1S115D and Ubc1S115A were rendered tolerant to thermal and reductive stress respectively. Ubc1 phosphorylation was higher in BY4741 cells compared to hog1Δ cells at 30 °C and was significantly reduced in BY4741 cells upon exposure at 40 °C. Taken together, the cell survival assays and Ubc1 phosphorylation status in strains and under conditions as described above suggest that tolerance to thermal and reductive stress in S. cerevisiae may be regulated by MAP kinase-mediated phosphorylation of Ubc1S97 and S115.
泛素结合酶 1（Ubc1）是 E2 酶家族的成员，它将泛素连接到注定通过泛素蛋白酶体系统降解的受损蛋白质上。它对于应激耐受是必要的，并且对于酿酒酵母的细胞存活是必不可少的。Ubc1 有五个丝氨酸残基，是激酶磷酸化的潜在底物。然而，没有数据表明 Ubc1 在酿酒酵母中的功能或应激耐受是由 Ubc1 的丝氨酸磷酸化调节的。我们证明 Ubc1 在丝氨酸残基上发生磷酸化。此外，在丝裂原激活蛋白（MAP）激酶丝氨酸残基（S97 和 S115）上表达“组成性磷酸化”或“去磷酸化”的 Ubc1 突变体，影响了酿酒酵母对热和还原应激的耐受。具体而言，Ubc1S97A 和 S115D 的表达增加了 BY4741 和 TetO7-UBC1ura3Δ 细胞的耐热性。在 40°C 下暴露后，BY4741 细胞中的 Ubc1 丝氨酸磷酸化减少。在相同菌株中，还原应激耐受与 Ubc1S97A 的表达相关。在类似条件下，Ubc1 磷酸化没有明显变化。表达 Ubc1S115D 和 Ubc1S115A 的 hog1Δ 和 slt2Δ 细胞分别对热和还原应激具有耐受性。与 hog1Δ 细胞相比，Ubc1 磷酸化在 30°C 时在 BY4741 细胞中更高，并且在 40°C 暴露时在 BY4741 细胞中显著降低。综上所述，上述菌株和条件下的细胞存活测定和 Ubc1 磷酸化状态表明，酿酒酵母对热和还原应激的耐受可能受 MAP 激酶介导的 Ubc1S97 和 S115 磷酸化调节。
