Schweikhard, Eva S. and Burckhardt, Birgitta C. and Joos, Friedericke and Fenollar-Ferrer, Cristina and Forrest, Lucy R. and Kempson, Stephen A. and Ziegler, Christine (2015) Role of N-glycosylation in renal betaine transport. BIOCHEMICAL JOURNAL, 470. pp. 169-179. ISSN 0264-6021, 1470-8728
Full text not available from this repository. (Request a copy)Abstract
The osmolyte and folding chaperone betaine is transported by the renal Na+ -coupled GABA (gamma-aminobutyric acid) symporter BGT-1 (betaine/GABA transporter 1), a member of the SLC6 (solute carrier 6) family. Under hypertonic conditions, the transcription, translation and plasma membrane (PM) insertion of BGT-1 in kidney cells are significantly increased, resulting in elevated betaine and GABA transport. Re-establishing isotonicity involves PM depletion of BGT-1. The molecular mechanism of the regulated PM insertion of BGT-1 during changes in osmotic stress is unknown. In the present study, we reveal a link between regulated PM insertion and N-glycosylation. Based on homology modelling, we identified two sites (Asn(171) and Asn(183)) in the extracellular loop 2 (EL2) of BGT-1, which were investigated with respect to trafficking, insertion and transport by immunogold-labelling, electron microscopy (EM), mutagenesis and two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radiolabelled substrate into MDCK (Madin-Darby canine kidney) and HEK293 (human embryonic kidney) cells. Trafficking and PM insertion of BGT-1 was clearly promoted by N-glycosylation in both oocytes and MDCK cells. Moreover, association with N-glycans at Asn(171) and Asn(183) contributed equally to protein activity and substrate affinity. Substitution of Asn(171) and Asn(183) by aspartate individually caused no loss of BGT-1 activity, whereas the double mutant was inactive, suggesting that N-glycosylation of at least one of the sites is required for function. Substitution by alanine or valine at either site caused a dramatic loss in transport activity. Furthermore, in MDCK cells PM insertion of N183D was no longer regulated by osmotic stress, highlighting the impact of N-glycosylation in regulation of this SLC6 transporter.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | NA+/MYO-INOSITOL COTRANSPORTER; SEROTONIN TRANSPORTER; HYPERTONIC STRESS; DOPAMINE TRANSPORTER; OSMOTIC REGULATION; NEUROTRANSMITTER TRANSPORTERS; FUNCTIONAL-CHARACTERIZATION; NOREPINEPHRINE TRANSPORTER; SCANNING MUTAGENESIS; LINKED GLYCOSYLATION; gamma-aminobutyric acid (GABA); transport; kidney; neurotransmitter:sodium symporter (NSS)/solute carrier 6 (SLC6) family; osmotic stress response; regulation; subcellular distribution |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Christine Ziegler |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 12 Jun 2019 12:59 |
| Last Modified: | 12 Jun 2019 12:59 |
| URI: | https://pred.uni-regensburg.de/id/eprint/4909 |
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