Browsing by Subject "osmotic stress response"

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    Role of N-glycosylation in renal betaine transport
    (Portland Press, 2015-08) Schweikhard, Eva S.; Burckhardt, Birgitta C.; Joos, Friedericke; Fenollar-Ferrer, Cristina; Forrest, Lucy R.; Kempson, Stephen A.; Ziegler, Christine; Department of Cellular & Integrative Physiology, IU School of Medicine
    The osmolyte and folding chaperone betaine is transported by the renal Na+-coupled GABA (γ-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 (Asn171 and Asn183) 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 Asn171 and Asn183 contributed equally to protein activity and substrate affinity. Substitution of Asn171 and Asn183 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.