Regulation of SLC26 proteins by plasma membrane recycling and novel interacting proteins

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Intestinal electroneutral NaCl absorption occurs by coupled Na/H- and Cl/HCO3-exchange mediated by NHE3 (SLC9A3) and DRA (down regulated in adenoma, SLC26A3). As for other SLC26 isoforms, regulation of their transport activity involves endocytosis/recycling resulting in varying numbers of transporters in the plasma membrane. DRA directly interacts with PDZ adapter proteins of the NHERF family (Na/H exchange regulatory factors 1-4 and sorting nexin 27; SNX27). We have shown that the PDZ interaction of DRA is important for its recycling. Preliminary work indicates that DRA interacts with SNX27 in the retromer complex in early endosomes. This suggests that other multi-protein complexes play an important role as well. Our aim is to better understand the intracellular trafficking of DRA, the related role of its PDZ interaction and the function of DRA-containing multi-protein complexes, and to apply this approach to other SLC26 isoforms. We will characterize DRA’s endocytic recycling in a time- and compartment-resolved manner using biochemical and microscopic techniques and applying an engineered extracellular tag. Fluorescent DRA-containing complexes will be monitored by blue native gel electrophoresis (BNE). Complexome profiling, which combines BNE and native mass spectrometry, and targeted interaction proteomics will reveal comprehensive information on protein complexes involved in DRA-internalization, endosomal trafficking and recycling. Cargo sorting proteins will be deciphered by deep proteomics and complexomics of immunoisolated DRA-containing endosomal fractions. Candidate proteins will be functionally characterized in HEK293 cells, differentiated intestinal Caco2 cells and intestinal organoids. We will further provide our proteomics platform to all members of the Research Unit FOR 5046 to study SLC26 complexes and functional consequences in proteome remodeling. The results will reveal molecular pathomechanisms involving SLC26 isoforms and allow future translational studies.