Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
Institute of Molecular Biology (IMB), Mainz, Germany.
Life Sci Alliance. 2021 Nov 11;5(2). doi: 10.26508/lsa.202000730. Print 2022 Feb.
N-terminal acetylation is a prominent protein modification, and inactivation of N-terminal acetyltransferases (NATs) cause protein homeostasis stress. Using multiplexed protein stability profiling with linear ubiquitin fusions as reporters for the activity of the ubiquitin proteasome system, we observed increased ubiquitin proteasome system activity in NatA, but not NatB or NatC mutants. We find several mechanisms contributing to this behavior. First, NatA-mediated acetylation of the N-terminal ubiquitin-independent degron regulates the abundance of Rpn4, the master regulator of the expression of proteasomal genes. Second, the abundance of several E3 ligases involved in degradation of UFD substrates is increased in cells lacking NatA. Finally, we identify the E3 ligase Tom1 as a novel chain-elongating enzyme (E4) involved in the degradation of linear ubiquitin fusions via the formation of branched K11, K29, and K48 ubiquitin chains, independently of the known E4 ligases involved in UFD, leading to enhanced ubiquitination of the UFD substrates.
N 端乙酰化是一种重要的蛋白质修饰，N 端乙酰转移酶（NATs）的失活会导致蛋白质稳态应激。我们使用多重蛋白质稳定性分析，通过线性泛素融合作为泛素蛋白酶体系统活性的报告，观察到 NatA 突变体中泛素蛋白酶体系统活性增加，而 NatB 或 NatC 突变体中没有。我们发现了几种促成这种行为的机制。首先，NatA 介导的 N 端非依赖泛素降解基序的乙酰化调节 Rpn4 的丰度，Rpn4 是蛋白酶体基因表达的主调控因子。其次，缺乏 NatA 的细胞中几种参与 UFD 底物降解的 E3 连接酶的丰度增加。最后，我们鉴定出 E3 连接酶 Tom1 是一种新型的链延伸酶（E4），通过形成分支的 K11、K29 和 K48 泛素链，参与线性泛素融合物的降解，不依赖于参与 UFD 的已知 E4 连接酶，导致 UFD 底物的泛素化增强。
