Seminar: Prof. Daniel Markovich

Structure, function and trafficking of epithelial sulfate anion transporters NaS1 and Sat1

Date:
14 January 2013 00:00 hrs.
Location:
Figdor Lecture Theatre, 8th floor RIMLS Building, Geert Grooteplein 26-28, route 289
Title:
Structure, function and trafficking of epithelial sulfate anion transporters NaS1 and Sat1
Speaker(s):

Prof. Daniel Markovich, Molecular Physiology Group, School of Biomedical Sciences, The University of Queensland, Australia.

Host(s):

Prof. René Bindels, Department of Physiology, NCMLS

14-01-2013 00:00:00Europe/AmsterdamStructure, function and trafficking of epithelial sulfate anion transporters NaS1 and Sat1 Figdor Lecture Theatre, 8th floor RIMLS Building, Geert Grooteplein 26-28, route 289Rimlsrimls@radboudumc.nl

Remarks / more information:

 

Markovich, DanielSulfate is an essential anion involved in many cellular processes. Cells obtain sulfate via sulfate transporters located on their plasma membranes. NaS1 (SLC13A1) and Sat1 (SLC26A1) are anion transporters that mediate renal proximal tubular and intestinal sulfate reabsorption and thereby regulate blood sulfate levels. Sat1 also mediates renal oxalate transport and controls blood oxalate levels. Targeted disruption of murine NaS1 and Sat1 leads to hyposulfatemia and hypersulfaturia. Sat1 null mice also exhibit hyperoxalemia, hyperoxaluria and calcium oxalate urolithiasis. NaS1 and Sat1 null mice also have other phenotypes that result due to changes in blood sulfate and oxalate levels. Experimental data indicates that NaS1 is essential for maintaining sulfate homeostasis, whereas Sat1 controls both sulfate and oxalate homeostasis in vivo. Despite their physiological roles, little is known about the structural identities of NaS1 and Sat1 and sorting mechanisms that regulate their cellular expression. NaS1 encodes a 595 amino acid 13 TMD protein localized to the apical (brush-border) membranes of epithelial cells. Sat1 encodes a 704 amino acid 12 TMD protein located on the basolateral membrane of cells. The aims of this study were to biochemically characterise the NaS1 and Sat1 proteins, identify the plasma membrane sorting mechanisms and determine the functional roles of non-synonymous SNPs in these proteins. Transient transfection of EGFP/NaS1 in renal epithelial cells (OK, LLC-PK1 and MDCK) demonstrated apical membrane expression, which was not affected by tunicamycin. Transfection of the EGFP/NaSi-1 N591S glycosylation mutant still led to apical expression, suggesting that apical sorting was independent of the glycosylation of this site. Treatment with cholesterol depleting compounds (lovastatin and methyl-&[beta]-cyclodextrin) was unable to disrupt apical sorting, suggesting that NaS1 apical trafficking may be independent of membrane lipid rafts. NaS1-His proteins analyzed by BN-PAGE appeared as a single complex. Dissociation revealed one additional band, indicating a dimeric structure of the complex. This data demonstrates that NaS1 forms a dimeric protein which is glycosylated at N591, whose sorting to the apical membrane in renal epithelial cells is independent of lipid rafts and glycosylation. Transfection of the Sat1 protein into MDCK and LLC-PK1 cells led to basolateral membrane sorting, as observed in vivo. To identify possible sorting determinants, truncations of the Sat1 cytoplasmic C-terminus were generated and transiently transfected into MDCK cells. Confocal microscopy revealed the removal of the last three residues on the Sat1 C-terminus, a putative PDZ domain, had no effect on the basolateral sorting in MDCK cells. Removal of the last 30 residues led to an intracellular expression for the GFP fusion protein suggesting a possible sorting motif lies between the last 3 and 30 residues of the Sat1 C-terminus. Elimination of a dileucine motif at position 677/678 resulted in the loss of basolateral sorting, suggesting this motif is required for Sat1 targeting to the basolateral membrane in kidney cells. Non-synonymous SNPs exist in the coding regions of human NaS1 (SLC13A1) and Sat1 (SLC26A1) genes. The functional relevance of these human SNPs and their effects on protein sorting will be described.



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