Novel regulatory mechanism of renaltubular transporter function by SUMOylation-deSUMOylation

SUMO（small ubiquitin-like protein）是泛素样蛋白的一种，通过与多种类似泛素系统的蛋白质如E1、E2、E3等以共价键结合而形成翻译后修饰系统。在本课题开始之前，本研究者已经发现SUMO-1和Ubc 9、E2 SUMO化酶与肾小管转运蛋白PEPT 2的相互作用，以及SUMO-1和皮亚斯、E3连接酶与转运蛋白TAT 1的相互作用。本研究的目的是通过SUMO化-去SUMO化和拮抗PDZ相互作用来阐明新型转运蛋白调控系统的分子机制。在这个项目中，我们研究了以下几点：1。阐明SUMO化相关蛋白与肾小管转运蛋白PEPT 2/TAT 1相互作用的分子机制;2.因此，首先，我们确定了PEPT 2/TAT 1细胞内C-末端SUMO化位点：我们使用在PEPT 2/TAT 1的C-末端的Lysin i position突变的克隆进行酵母双杂交研究SUMO化的共同序列KXD/E（ε：疏水性氨基酸残基），以证实其重要性。我们发现这些突变体失去了相互作用，表明这些位点是结合所必需的。第二，我们观察了在基因过表达的MDCK细胞中使用GFP融合的PEPT 2/TAT 1全长蛋白的PEPT 2/TAT 1的细胞内定位。转染这些克隆后，我们可以通过共聚焦激光扫描显微镜证实TAT 1蛋白的基底侧表达。

SUMO (small ubiquitin-like protein), one of the ubiquitin-like proteins, functions as a posttranslational modification system by binding with various proteins with covalent bond similar to ubiquitin system such as E1, E2, E3. Before starting this project, this researcher had already found the interaction of SUMO-1 and Ubc9, E2 SUMOylation enzyme, with renal tubular transporter PEPT2 and that of SUMO-1 and PIAS, E3 ligase with transporter TAT1. The purpose of this study was to clarify the molecular mechanism of novel regulatory system for transporters by SUMOylation-deSUMOylation and antagonism to PDZ interaction. In this project, we examined the followings:1. Clarification of molecular mechanism for the interaction of SUMOylation-related proteins with renal tubular transporters PEPT2/TAT1;2. Effects of PDZ interaction via intracellular C-termini of PEPT2/TAT1on SUMOylation.As a result, first, we identified the sites of SUMOylation in the intracellular C-termini of PEPT2/TAT1：we performed the yeast two-hybrid studies using the clones that have mutation of Lysin iposition n SUMOylation concensus sequence ψKXD/E（ψ: hydrophobic amino acid residue）in C-termini of PEPT2/TAT1 to confirm its importance. We found that those mutants lost the interaction indicating that those sites are necessary for the bindings. Second, we observed the intracellular localization of PEPT2/TAT1 using GFP-fused PEPT2/TAT1 full-length proteins in gene-overexpressed MDCK cells. After the transfection of those clones, we could confirm the basolateral expression of TAT1 protein by confocal laser-scanning microscopy.