Novel regulatory mechanism of renaltubular transporter function by SUMOylation-deSUMOylation

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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.