Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara, Japan.
Biosci Biotechnol Biochem. 2021 Apr 24;85(5):1266-1274. doi: 10.1093/bbb/zbab028.
Most of plasma membrane transporters are downregulated by ubiquitination-dependent endocytosis to avoid the excess uptake of their substrates. In Saccharomyces cerevisiae, ubiquitination of transporters is mediated by the HECT-type ubiquitin ligase Rsp5. We report here a mechanism underlying the substrate-induced endocytosis of the broad-specificity amino acid permease Agp1. First, we found that Agp1 underwent ubiquitination and endocytosis in response to the addition of excess asparagine, which is a substrate of Agp1. Moreover, the substrate-induced internalization of Agp1 was dependent on the ubiquitination activity of Rsp5. Since Rsp5 requires α-arrestin family proteins as adaptors to bind with substrates, we next developed a method of genetic screening to identify adaptor proteins for Agp1 endocytosis. This screening and biochemical analysis revealed that Bul1, but not its paralogue Bul2, was essential for the substrate-induced endocytosis of Agp1. Our results support that the substrate-induced endocytosis of Agp1 requires Rsp5 and Bul1.
大多数质膜转运蛋白通过泛素化依赖的内吞作用被下调，以避免其底物的过度摄取。在酿酒酵母中，转运蛋白的泛素化由 HECT 型泛素连接酶 Rsp5 介导。我们在此报告了广谱氨基酸渗透酶 Agp1 的底物诱导内吞作用的机制。首先，我们发现 Agp1 在过量天冬酰胺（Agp1 的底物）存在的情况下发生泛素化和内吞作用。此外，Agp1 的底物诱导内吞作用依赖于 Rsp5 的泛素化活性。由于 Rsp5 需要 α-抑制蛋白家族蛋白作为衔接蛋白与底物结合，我们接下来开发了一种遗传筛选方法来鉴定 Agp1 内吞作用的衔接蛋白。该筛选和生化分析表明，Bul1（而非其同源物 Bul2）对于 Agp1 的底物诱导内吞作用是必需的。我们的结果支持 Agp1 的底物诱导内吞作用需要 Rsp5 和 Bul1。
