کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1985124 | 1539969 | 2007 | 12 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Quaternary structure and apical membrane sorting of the mammalian NaSi-1 sulfate transporter in renal cell lines
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کلمات کلیدی
eGFPBN-PAGEMDCKLLC-PK1N-glycosylation - N-گلیکوزیلتblue native-polyacrylamide gel electrophoresis - الکتروفورز ژل آبی بومی پلی آکریل آمیدXenopus laevis oocytes - تخمک Xenopus laevisOpossum kidney cells - سلول های کلیوی اپوسومMadin-Darby canine kidney cells - سلول های کلیوی کانین Madin-DarbySorting - مرتب سازیenhanced green fluorescence protein - پروتئین فلورسانس سبز افزایش یافته است
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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 75Â kDa. PNGase F treatment shifted both bands to 57Â 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: The International Journal of Biochemistry & Cell Biology - Volume 39, Issue 12, 2007, Pages 2240-2251
Journal: The International Journal of Biochemistry & Cell Biology - Volume 39, Issue 12, 2007, Pages 2240-2251
نویسندگان
Ralf R. Regeer, Annette Nicke, Daniel Markovich,