Quaternary structure and apical membrane sorting of the mammalian NaSi-1 sulfate transporter in renal cell lines.
NaSi-1 encodes a Na(+)-sulfate cotransporter expressed on the apical membrane of renal proximal tubular cells, which is responsible for body sulfate homeostasis. Limited information is available on NaSi-1 protein structure and the mechanisms controlling its apical membrane sorting. The aims of this study were to biochemically determine the quaternary structure of the rat NaSi-1 protein and to characterize its expression in renal epithelial cell lines. Hexahistidyl-tagged NaSi-1 (NaSi-1-His) proteins expressed in Xenopus oocytes, appeared as two bands of about 60 and 75 kDa. PNGase F treatment shifted both bands to 57 kDa while endoglycosidase H treatment led to a downward shift of the lower molecular mass band only. Mutagenesis of a putative N-glycosylation site (N591S) produced a single band that was not shifted by endoglycosidase H or PNGase F, confirming a single glycosylation site at residue 591. Blue native-PAGE and cross-linking experiments revealed dimeric complexes, suggesting the native form of NaSi-1 to be a dimer. Transient transfection of EGFP/NaSi-1 in renal epithelial cells (OK, LLC-PK1 and MDCK) demonstrated apical membrane sorting, which was insensitive to tunicamycin. Transfection of the EGFP/NaSi-1 N591S glycosylation mutant also showed apical expression, suggesting N591 is not essential for apical sorting. Treatment with cholesterol depleting compounds did not disrupt apical sorting, but brefeldin A led to misrouting to the basolateral membrane, suggesting that NaSi-1 sorting is through the ER to Golgi pathway. Our data demonstrates that NaSi-1 forms a dimeric protein which is glycosylated at N591, whose sorting to the apical membrane in renal epithelial cells is brefeldin A-sensitive and independent of lipid rafts or glycosylation.
Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, St. Lucia, Brisbane, Qld 4072, Australia.
Ralf R
RR
Regeer
Annette
A
Nicke
Daniel
D
Markovich
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D004229:Dithiothreitol / Q000737:chemistry; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D009893:Opossums; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D000077003:Sodium Sulfate Cotransporter; D013552:Swine; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D014508:Urea / Q000737:chemistry; D014982:Xenopus laevis
D000924:Anticholesteremic Agents; D027682:Cation Transport Proteins; D003902:Detergents; D000077003:Sodium Sulfate Cotransporter; D027981:Symporters; D014415:Tunicamycin; D020126:Brefeldin A; D014508:Urea; D004229:Dithiothreitol