Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara, 630-0192, Japan.
Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara, 630-0192, Japan.
Biochem Biophys Res Commun. 2020 Oct 20;531(3):416-421. doi: 10.1016/j.bbrc.2020.07.117. Epub 2020 Aug 14.
Proline is one of the abundant amino acids in grape must, but in winemaking processes it is poorly assimilated by the yeast Saccharomyces cerevisiae. This often causes a nitrogen deficiency during fermentation and proline accumulation in wine. Our previous study showed that arginine inhibits proline utilization by specifically inducing the endocytosis of the high-affinity proline transporter Put4. However, the detailed mechanisms underlying this induction are still unclear. Here, we propose a possible mechanism mediated by the ubiquitin ligase Rsp5 and its adaptor protein, Art3. First, we found that the ubiquitination activity of Rsp5 was essential for the arginine-induced endocytosis of Put4. Because Put4 contains no Rsp5-binding motif, we next screened an adaptor protein that plays a role in the arginine-induced endocytosis of Put4. Our genetic and biochemical analyses clearly revealed that the ART3 gene-disrupted cells were defective in Put4 endocytosis, indicating that Art3 is a key regulator for Put4 endocytosis. More importantly, we discovered that deletion of ART3 remarkably canceled the inhibitory effects of arginine on proline utilization. The present results could hold promise for the development of wine yeast strains that can efficiently assimilate the abundant proline in grape must during the fermentation processes.
脯氨酸是葡萄汁中含量丰富的氨基酸之一，但在酿酒过程中，其被酿酒酵母（Saccharomyces cerevisiae）吸收较差。这通常会导致发酵过程中氮缺乏和葡萄酒中脯氨酸积累。我们之前的研究表明，精氨酸通过特异性诱导高亲和力脯氨酸转运蛋白 Put4 的内吞作用来抑制脯氨酸的利用。然而，这种诱导的详细机制仍不清楚。在这里，我们提出了一种可能的机制，该机制由泛素连接酶 Rsp5 和其衔接蛋白 Art3 介导。首先，我们发现 Rsp5 的泛素化活性对于精氨酸诱导的 Put4 内吞作用是必需的。由于 Put4 不含 Rsp5 结合基序，因此我们接下来筛选了在精氨酸诱导的 Put4 内吞作用中起作用的衔接蛋白。我们的遗传和生化分析清楚地表明，Art3 基因缺失细胞的 Put4 内吞作用受损，表明 Art3 是 Put4 内吞作用的关键调节剂。更重要的是，我们发现 Art3 的缺失显着取消了精氨酸对脯氨酸利用的抑制作用。这些结果可能为开发在发酵过程中能够有效吸收葡萄汁中丰富脯氨酸的葡萄酒酵母菌株提供了希望。
