Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Mol Biol Cell. 2013 Apr;24(7):890-900. doi: 10.1091/mbc.E12-11-0838. Epub 2013 Jan 30.
Two conserved ubiquitin ligases, Hrd1 and Doa10, mediate most endoplasmic reticulum-associated protein degradation (ERAD) in yeast. Degradation signals (degrons) recognized by these ubiquitin ligases remain poorly characterized. Doa10 recognizes the Deg1 degron from the MATα2 transcription factor. We previously found that deletion of the gene (NAT3) encoding the catalytic subunit of the NatB N-terminal acetyltransferase weakly stabilized a Deg1-fusion protein. By contrast, a recent analysis of several MATα2 derivatives suggested that N-terminal acetylation of these proteins by NatB was crucial for recognition by Doa10. We now analyze endogenous MATα2 degradation in cells lacking NatB and observe minimal perturbation relative to wild-type cells. However, NatB mutation strongly impairs degradation of ER-luminal Hrd1 substrates. This unexpected defect derives from a failure of Der1, a Hrd1 complex subunit, to be N-terminally acetylated in NatB mutant yeast. We retargeted Der1 to another acetyltransferase to show that it is the only ERAD factor requiring N-terminal acetylation. Preventing Der1 acetylation stimulates its proteolysis via the Hrd1 pathway, at least partially accounting for the ERAD defect observed in the absence of NatB. These results reveal an important role for N-terminal acetylation in controlling Hrd1 ligase activity toward a specific class of ERAD substrates.
两种保守的泛素连接酶，Hrd1 和 Doa10，介导酵母中大多数内质网相关蛋白降解（ERAD）。这些泛素连接酶识别的降解信号（degrons）仍然知之甚少。Doa10 识别 MATα2 转录因子的 Deg1 降解信号。我们之前发现，编码 NatB N 端乙酰转移酶催化亚基的基因（NAT3）缺失会轻微稳定 Deg1 融合蛋白。相比之下，最近对几种 MATα2 衍生物的分析表明，NatB 对这些蛋白质的 N 端乙酰化对于被 Doa10 识别至关重要。我们现在分析了缺乏 NatB 的细胞中内源性 MATα2 的降解情况，与野生型细胞相比几乎没有受到干扰。然而，NatB 突变强烈损害了 ER 腔 Hrd1 底物的降解。这个意外的缺陷源自于 NatB 突变酵母中 Hrd1 复合物亚基 Der1 的 N 端未能乙酰化。我们将 Der1 重新靶向到另一个乙酰转移酶上，以证明它是唯一需要 N 端乙酰化的 ERAD 因子。防止 Der1 乙酰化通过 Hrd1 途径刺激其蛋白水解，至少部分解释了在缺乏 NatB 时观察到的 ERAD 缺陷。这些结果揭示了 N 端乙酰化在控制 Hrd1 连接酶对特定类别的 ERAD 底物的活性方面的重要作用。
