The kidney plays a central role in fluid and electrolyte homeostasis as well as for the excretion of xenobiotics. For this purpose, a multitude of transporters and other transmembrane proteins is expressed along nephrons with high density and distinct cellular localization. Peptides, metabolites and inorganic ions are mainly resorbed in the proximal tubules by epithelial cells carrying tightly packed microvilli (together forming the brush border). These membrane structures are densely packed with transporters, but how these are molecularly organized and functionally cooperate is largely unknown.

To resolve the molecular composition of renal transporters we combined two proteomic approaches: (i) we used high-resolution complexome profiling (based on cryo-slicing Blue Native Gel mass spectrometry (csBN-MS); Müller et al., 2016, Schulte et al., 2023) to analyse a preparation of brush border-enriched membranes from mouse kidney. After native separation of gently solubilized protein complexes, the respective gel was cut into 280 slices à 0.3 mm which were tryptically digested and comprehensively analyzed by LC-MS/MS on a Orbitrap Exploris mass spectrometer. Proteins were identified with PEAKS and quantified using an elaborate in-house developed processing and profile building pipeline (Schulte et al., 2023). (ii) The sodium-phosphate transporter SLC34A1 (NPT2A) and other selected examples of newly identified transporter complexes were further investigated by multi-epitope affinity purification mass spectrometry (meAP-MS), i.e. using multiple target-specific antibodies in wildtype and target knockout kidney membrane solubilisates as well as target-unrelated antibody controls.

Complexome profiling analysis provided more than 5000 detailed abundance-size distributions of proteins with strong representation of bona-fide brush-border transmembrane transporters. Inspection of the profile peaks revealed that the majority of transmembrane proteins are not existing as monomers but are assembled into

several higher molecular weight complexes (as in Schulte et al., 2023). Apart from known homo- and heteromeric transporter complexes, a number of peak correlations indicated the formation of novel transporter supercomplexes and assembly of transporters with yet unknown subunits. These results were corroborated by subsequent meAP-MS analyses. Thus, the obtained NPT2A interactome comprised the known interaction partners PDZ1L and NHRF3, in addition to 20 proteins that had not yet been associated with sodium-phosphate transporters and their cell biology. Most notably, NPT2A was found to interact with a subset of transporters coincident with high-molecular weight peak clusters associated with NPT2A in the brush border complexome.

The functional and structural implications of these findings are currently investigated by electrophysiological experiments (upon heterologous reconstitution) and ultrastructural methods (like freeze-fracture immuno-electron microscopy).