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 truncat...
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Nature Portfolio
2018
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oai:doaj.org-article:88514809b5334da8bc2989f2186a830f2021-12-02T15:08:49ZTrafficking, localization and degradation of the Na+,HCO3 − co-transporter NBCn1 in kidney and breast epithelial cells10.1038/s41598-018-25059-72045-2322https://doaj.org/article/88514809b5334da8bc2989f2186a830f2018-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-25059-7https://doaj.org/toc/2045-2322Abstract 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.Christina Wilkens OlesenJens VogensenIda AxholmMarc SeverinJulie SchnipperIsabella Skandorff PedersenJakob Hjorth von StemannJacob Morville SchrøderDan Ploug ChristensenStine Falsig PedersenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-16 (2018) |
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Medicine R Science Q Christina Wilkens Olesen Jens Vogensen Ida Axholm Marc Severin Julie Schnipper Isabella Skandorff Pedersen Jakob Hjorth von Stemann Jacob Morville Schrøder Dan Ploug Christensen Stine Falsig Pedersen Trafficking, localization and degradation of the Na+,HCO3 − co-transporter NBCn1 in kidney and breast epithelial cells |
| description |
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. |
| format |
article |
| author |
Christina Wilkens Olesen Jens Vogensen Ida Axholm Marc Severin Julie Schnipper Isabella Skandorff Pedersen Jakob Hjorth von Stemann Jacob Morville Schrøder Dan Ploug Christensen Stine Falsig Pedersen |
| author_facet |
Christina Wilkens Olesen Jens Vogensen Ida Axholm Marc Severin Julie Schnipper Isabella Skandorff Pedersen Jakob Hjorth von Stemann Jacob Morville Schrøder Dan Ploug Christensen Stine Falsig Pedersen |
| author_sort |
Christina Wilkens Olesen |
| title |
Trafficking, localization and degradation of the Na+,HCO3 − co-transporter NBCn1 in kidney and breast epithelial cells |
| title_short |
Trafficking, localization and degradation of the Na+,HCO3 − co-transporter NBCn1 in kidney and breast epithelial cells |
| title_full |
Trafficking, localization and degradation of the Na+,HCO3 − co-transporter NBCn1 in kidney and breast epithelial cells |
| title_fullStr |
Trafficking, localization and degradation of the Na+,HCO3 − co-transporter NBCn1 in kidney and breast epithelial cells |
| title_full_unstemmed |
Trafficking, localization and degradation of the Na+,HCO3 − co-transporter NBCn1 in kidney and breast epithelial cells |
| title_sort |
trafficking, localization and degradation of the na+,hco3 − co-transporter nbcn1 in kidney and breast epithelial cells |
| publisher |
Nature Portfolio |
| publishDate |
2018 |
| url |
https://doaj.org/article/88514809b5334da8bc2989f2186a830f |
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1718388010879287296 |