The Na+;HCO3− co-transporter NBCn1 (SLC4A7) is a major regulator of intracellular pH yet its trafficking and turnover are essentially unstudied. Here, we used MDCK-II and MCF-7 cells to investigate these processes in epithelial cells. GFP-NBCn1 membrane localization was abolished by truncation of the full NBCn1 C-terminal tail (C-tail) yet did not require the C-terminal PDZ-binding motif (ETSL). Glutathione-S-Transferase-pulldown of the C-tail followed by mass spectrometry analysis revealed putative interactions with multiple sorting-, degradation- and retention factors, including the scaffolding protein RACK1. Pulldown of FLAG-tagged deletion constructs mapped the RACK1 interaction to the proximal NBCn1 C-tail. Proximity Ligation Assay and co-immunoprecipitation confirmed that native NBCn1 interacts with RACK1 in a cellular context. Consistent with a functional role of this complex, RACK1 knockdown reduced NBCn1 membrane localization without affecting total NBCn1 expression. Notably, only non-confluent cells exhibited detectable NBCn1-RACK1 plasma membrane co-localization, suggesting that RACK1 regulates the trafficking of NBCn1 to the membrane. Whereas total NBCn1 degradation was slow, with a half-life of more than 24 h, one-third of surface NBCn1 was constitutively endocytosed from the basolateral membrane within 60 min. This suggests that a fraction of NBCn1 exhibits recycling between the basolateral membrane and intracellular compartment(s). Our findings have important implications for understanding NBCn1 regulation as well as its dysregulation in disease.

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.

We previously showed that the Na+,HCO3‐ cotransporter NBCn1 (SLC4A7) is upregulated in breast cancer, and that cisplatin treatment of breast cancer cells causes pronounced loss of NBCn1 plasma membrane localization. However, essentially nothing is known regarding the mechanisms of NBCn1 trafficking and turnover. NBCn1 localized predominantly to the basolateral membrane in polarized breast cancer cells, with a highly cell‐density dependent expression profile. Removal of the cytosolic NBCn1 C‐terminal tail (NBCn1‐Ct) strongly reduced NBCn1 membrane localization in HEK293 cells. GST pull‐down in MCF‐7 cells followed by LTQ Orbitrap Mass spectrometry identified Receptor C kinase‐1 (RACK1), retromer complex components vacuolar protein sorting‐associated protein 35 (VPS35) and Sorting Nexin 27 (SNX27), and AP1 and ‐2 adaptor proteins, as NBCn1‐Ct interaction partners. Co‐immunoprecipitation and immunofluorescence analysis confirmed interaction between RACK1 and NBCn1 in MCF‐7 cells. Furthermore, siRNA‐mediated RACK1 knockdown reduced NBCn1 expression and strongly attenuated its plasma membrane localization. SNX27 and VPS35 partially colocalized with NBCn1 in breast cancer cell lines, in a punctate pattern closely adjacent to the plasma membrane, suggestive of a role in NBCn1 trafficking. In conclusion, these preliminary findings point to the involvement of RACK1 and likely also VPS35 and SNX27, in regulation of NBCn1 expression and localization in breast cancer cells.