Johnson Daniel L, Kumar Ravinder, Kakhniashvili David, Pfeffer Lawrence M, Laribee R Nicholas
Molecular Bioinformatics Core and the University of Tennessee Health Science Center Office of Research, University of Tennessee Health Science Center, Memphis, TN, United States of America.
Department of Pathology and Laboratory Medicine, College of Medicine and the Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN, United States of America.
bioRxiv. 2023 Aug 28:2023.08.28.555095. doi: 10.1101/2023.08.28.555095.
The Ccr4-Not complex containing the Not4 ubiquitin ligase regulates gene transcription and mRNA decay, yet it also has poorly defined roles in translation, proteostasis, and endolysosomal-dependent nutrient signaling. To define how Ccr4-Not mediated ubiquitin signaling regulates these additional processes, we performed quantitative proteomics in the yeast lacking the Not4 ubiquitin ligase, and also in cells overexpressing either wild-type or functionally inactive ligase. Herein, we provide evidence that both increased and decreased Ccr4-Not ubiquitin signaling disrupts ribosomal protein (RP) homeostasis independently of reduced RP mRNA changes or reductions in known Not4 ribosomal substrates. Surprisingly, we also find that both Not4-mediated ubiquitin signaling, and the Ccr4 subunit, actively inhibit 40S ribosomal autophagy. This 40S autophagy is independent of canonical Atg7-dependent macroautophagy, thus indicating microautophagy activation is responsible. Furthermore, the Not4 ligase genetically interacts with endolysosomal pathway effectors to control both RP expression and 40S autophagy efficiency. Overall, we demonstrate that balanced Ccr4-Not ligase activity maintains RP homeostasis, and that Ccr4-Not ubiquitin signaling interacts with the endolysosomal pathway to both regulate RP expression and inhibit 40S ribosomal autophagy.
包含Not4泛素连接酶的Ccr4-Not复合物可调节基因转录和mRNA降解，但它在翻译、蛋白质稳态和内溶酶体依赖性营养信号传导中的作用仍不明确。为了确定Ccr4-Not介导的泛素信号如何调节这些额外的过程，我们在缺乏Not4泛素连接酶的酵母中，以及在过表达野生型或功能失活连接酶的细胞中进行了定量蛋白质组学研究。在此，我们提供证据表明，Ccr4-Not泛素信号的增加和减少都会破坏核糖体蛋白（RP）的稳态，这与RP mRNA变化减少或已知Not4核糖体底物减少无关。令人惊讶的是，我们还发现Not4介导的泛素信号和Ccr4亚基都会积极抑制40S核糖体自噬。这种40S自噬独立于经典的Atg7依赖性巨自噬，因此表明微自噬激活是其原因。此外，Not4连接酶与内溶酶体途径效应器发生遗传相互作用，以控制RP表达和40S自噬效率。总体而言，我们证明平衡的Ccr4-Not连接酶活性可维持RP稳态，并且Ccr4-Not泛素信号与内溶酶体途径相互作用，以调节RP表达并抑制40S核糖体自噬。
