School of Biosciences, University of Birmingham, Edgbaston, West Midlands, B15 2TT, UK.
CEA, CNRS, Université Paris-Saclay, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, 91198, France.
Plant Physiol. 2023 Oct 26;193(3):2086-2104. doi: 10.1093/plphys/kiad406.
The acetylation-dependent (Ac/)N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3 ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multiomics approaches to characterize potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3 ligases in the Nt-acetylation-(NTA)-dependent turnover of proteins at global- and protein-specific scales. Arabidopsis has two endoplasmic reticulum (ER)-localized DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wild type, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localized SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.
乙酰化依赖性（Ac/）N-肽段途径通过 E3 连接酶（称为 Ac/N-识别蛋白）识别其乙酰化的 N 末端（Nt）来降解蛋白质。迄今为止，植物中尚未定义特定的 Ac/N-识别蛋白。在这里，我们使用分子、遗传和多组学方法来表征拟南芥（Arabidopsis thaliana）DEGRADATION OF ALPHA2 10（DOA10）-样 E3 连接酶在蛋白质 Nt-乙酰化（NTA）依赖性全局和蛋白质特异性水平上的作用。拟南芥有两个内质网（ER）定位的 DOA10 样蛋白。AtDOA10A，但不是 Brassicaceae 特异性的 AtDOA10B，可以弥补酵母（Saccharomyces cerevisiae）ScDOA10 功能的丧失。Atdoa10a/b RNAi 突变体的转录组和 Nt-乙酰组谱分析显示，与野生型相比，全局 NTA 谱没有明显差异，这表明 AtDOA10s 不调节 NTA 底物的批量周转。在酵母和拟南芥中使用蛋白质稳态和环己酰亚胺追踪降解测定，我们表明，ER 定位的鲨烯环氧化酶 1（AtSQE1），一种关键的固醇生物合成酶的周转，是由 AtDOA10s 介导的。AtSQE1 在植物体内的降解不依赖于 NTA，但 Nt-乙酰转移酶间接影响其在酵母中的周转，这表明 NTA 和细胞蛋白稳态在王国之间存在差异。我们的工作表明，与酵母和哺乳动物不同，Nt-乙酰化蛋白的靶向不是拟南芥 DOA10 样 E3 连接酶的主要功能，并进一步深入了解植物 ERAD 和控制真核生物固醇生物合成的调节机制的保守性。
