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.
Highlights
The electroneutral Na+;HCO3− co-transporter NBCn1 (SLC4A7) is a member of the SLC4 family of bicarbonate transport proteins and is a major mediator of net cellular acid extrusion in most tissues studied[1,2]
Native NBCn1 localizes to the basolateral membrane in polarized Madin-Darby Canine Kidney II (MDCK-II) and MCF-7 cells
Zona occludens protein 1 (ZO-1) and E-cadherin were used as markers of tight junctions and of the basolateral domain, respectively[29]
Summary
The electroneutral Na+;HCO3− co-transporter NBCn1 (SLC4A7) is a member of the SLC4 family of bicarbonate transport proteins and is a major mediator of net cellular acid extrusion in most tissues studied[1,2]. In some parts of the choroid plexus, NBCn1 is found at the apical membrane[23], and truncation of the entire C-terminal was found to abolish NBCn1 membrane targeting in HEK293 cells[24]. It appears that NBCn1 sorting mechanisms differ between different epithelial cells, and that at least part of the signal(s) for membrane targeting reside in the C-terminal. PDZ-binding motifs play important roles in sorting and retention of membrane proteins in epithelial cells[25], yet truncation of the PDZ-binding motif did not attenuate plasma membrane NBCn1 activity in HEK293 cells[26]
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