Microbial Molecular Genetics Laboratory, Institute of Biosciences and Applications, National Centre for Scientific Research "Demokritos", Patr. Grigoriou E & 27 Neapoleos St., 15341 Agia Paraskevi, Greece.
Molecular Physiology of the Cell Laboratory, Université Libre de Bruxelles (ULB), IBMM, 6041 Gosselies, Belgium.
Int J Mol Sci. 2021 Sep 22;22(19):10208. doi: 10.3390/ijms221910208.
Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.
选择性内吞作用随后降解是下调质膜转运蛋白以响应特定环境信号的主要机制。在此过程中，内吞作用是由 Rsp5 泛素连接酶催化的泛素化促进的，通过 α-抑制蛋白家族的衔接蛋白靶向转运蛋白。然而，这种靶向的分子机制及其根据条件的控制仍不完全清楚。在这项工作中，我们剖析了引发精氨酸渗透酶 Can1 内吞的分子机制，以响应环己酰亚胺诱导的 TORC1 过度激活。我们表明，环己酰亚胺以依赖 Bul1/2α-抑制蛋白的方式促进 Rsp5 依赖性 Can1 泛素化和内吞作用。对于这种下调，Can1 N 端的短酸性补丁序列也至关重要，该序列可能作为 Bul1/2 的结合位点。先前报道的环己酰亚胺抑制转运蛋白从反式高尔基体网络到质膜的回收，似乎也有助于有效的 Can1 下调。我们的结果还表明，与先前描述的由 Art1α-抑制蛋白介导的、由底物转运引发的 Can1 内吞作用相反，Bul1/2 介导的 Can1 泛素化发生与转运蛋白的构象无关。这项研究进一步深入了解了不同的α-抑制蛋白如何在不同条件下控制特定氨基酸转运蛋白的依赖泛素的下调。
