Trafficking, localization and degradation of the Na+,HCO3− co-transporter NBCn1 in kidney and breast epithelial cells

Abstract

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.

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells

“…Role (s) for previously described cell cycle regulatory signaling pathways [40] seems likely, however, also the pH changes during the cell cycle could be involved. For example, ChIP-seq analysis has shown that the NHE1 promoter interacts with, among many others, the transcription factors E2F1 and Egr-1 [60]. E2F1 is a major regulator of cell cycle dependent transcription [40], and it is interesting to note that its expression profile as shown in this study fits well with the NHE1 expression profile during the cell cycle (compare Figures 4(a) and 6(b)).…”

Dynamic subcellular localization of sodium-bicarbonate cotransporter NBCn1/SLC4A7 to plasma membrane, centrosomes, spindle, and primary cilia

“…As we predicted from our RACK1 interaction analysis (Olesen et al, 2018), truncation of the entire C-terminal distal to α-helix I (i.e. 1139-1254) had no effect on NBCn1 plasma membrane expression (Fig.…”

Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival

“…Whereas the short delay from tissue isolation to functional evaluation is a unique strength of the current study—because it minimizes the risk of phenotypical changes and thereby strengthens the connection to the clinical condition—it limits the experimental possibilities for detailed mechanistic and molecular studies. The experimental setup allows for manipulation and precise control of the buffer compositions; but with half-lives of protein degradation of 76 and 48 hr for NBCn1 and NHE1, respectively ( Olesen et al, 2018 ), we cannot realistically reduce overall cellular protein levels by interfering with their expression—e.g., by RNAi knockdown technologies ( Boedtkjer et al, 2006 )—in the human biopsy material. Also, the available pharmacological options are too unspecific and without selectivity for individual Na + ,HCO 3 – cotransporters ( Boedtkjer et al, 2016 ; Boedtkjer et al, 2012 ; Larsen et al, 2012 ).…”