Trafficking, localization and degradation of the Na+,HCO3- Co-transporter NBCn1 in kidney and breast epithelial cells
| Autor: | Julie Schnipper, Marc Severin, Isabella Skandorff Pedersen, Christina W. Olesen, Dan Ploug Christensen, Ida Axholm, Stine F. Pedersen, Jens Vogensen, Jakob Hjorth von Stemann, Jacob M. Schrøder |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Scaffold protein Intracellular pH Science Proximity ligation assay Kidney Article Madin Darby Canine Kidney Cells 03 medical and health sciences 0302 clinical medicine Dogs Animals Humans Amino Acid Sequence Breast Epithelial polarity Gene knockdown Multidisciplinary Chemistry Sodium-Bicarbonate Symporters Cell Membrane Transporter Epithelial Cells Transport protein Cell biology Kinetics Protein Transport 030104 developmental biology 030220 oncology & carcinogenesis Proteolysis MCF-7 Cells Medicine Lysosomes Intracellular |
| Zdroj: | Olesen, C W, Vogensen, J, Axholm, I, Severin, M, Schnipper, J, Pedersen, I S, von Stemann, J H, Schrøder, J M, Christensen, D P & Pedersen, S F 2018, ' Trafficking, localization and degradation of the Na +,HCO 3-Co-transporter NBCn1 in kidney and breast epithelial cells ', Scientific Reports, vol. 8, 7435, pp. 1-16 . https://doi.org/10.1038/s41598-018-25059-7 Scientific Reports Scientific Reports, Vol 8, Iss 1, Pp 1-16 (2018) |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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