Role of N-glycosylation in renal betaine transport.
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 (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.
Structural Biology Department Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, 60438 Frankfurt am Main, Germany Faculty of Biology and Preclinical Medicine, Universitätsstraße 31, 93040 Regensburg, Germany.
Eva S
ES
Schweikhard
Department of Systemic Physiology and Pathophysiology, University Medical Center Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.
Birgitta C
BC
Burckhardt
Structural Biology Department Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, 60438 Frankfurt am Main, Germany.
Friedericke
F
Joos
Computational Structural Biology Group, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
Cristina
C
Fenollar-Ferrer
Computational Structural Biology Group, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
Lucy R
LR
Forrest
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, U.S.A.
Stephen A
SA
Kempson
Structural Biology Department Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, 60438 Frankfurt am Main, Germany Faculty of Biology and Preclinical Medicine, Universitätsstraße 31, 93040 Regensburg, Germany Christine.Ziegler@biophys.mpg.de.
Christine
C
Ziegler
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001622:Betaine / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D009997:Osmotic Pressure; D011134:Polysaccharides / Q000378:metabolism; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D011134:Polysaccharides; C072207:betaine plasma membrane transport proteins; D001224:Aspartic Acid; D001622:Betaine; D005680:gamma-Aminobutyric Acid; D001216:Asparagine
kidney; neurotransmitter:sodium symporter (NSS)/solute carrier 6 (SLC6) family; osmotic stress response; regulation; subcellular distribution; transport; γ-aminobutyric acid (GABA)