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.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Olesen CW,Vogensen J,Axholm I,Severin M,Schnipper J,Pedersen IS,von Stemann JH,Schrøder JM,Christensen DP,Pedersen SF

doi

10.1038/s41598-018-25059-7

subject

Has Abstract

pub_date

2018-05-09 00:00:00

pages

7435

issue

1

issn

2045-2322

pii

10.1038/s41598-018-25059-7

journal_volume

8

pub_type

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