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Studies on the topology of the renal type II NaPi-cotransporter.
Journal article

Studies on the topology of the renal type II NaPi-cotransporter.

  • 1999-06-17
Published in:
  • Pflugers Archiv : European journal of physiology. - 1999
Amino Acid Sequence
Animals
Carrier Proteins
Cell Line
Dogs
Epitopes
Female
Glycosylation
Immunohistochemistry
Immunosorbent Techniques
Kidney Tubules, Proximal
Microvilli
Molecular Sequence Data
Oligopeptides
Oocytes
Peptide Fragments
Peptides
Rats
Sodium-Phosphate Cotransporter Proteins
Sodium-Phosphate Cotransporter Proteins, Type II
Symporters
Transfection
Xenopus laevis
English The rat type II sodium/phosphate cotransporter (NaPi-2) is a 85- to 90-kDa glycosylated protein located at the proximal tubular brush border membrane. Hydropathy predictions suggest eight transmembrane domains (sTM) with a large glycosylated loop between sTM 3 and sTM 4. We have studied the membrane topology of NaPi-2 expressed in oocytes. A 33-amino-acid fragment containing the FLAG epitope was inserted into seven loops connecting the sTMs and into the NH2- and COOH-ends of the protein. FLAG-antibody binding suggested that the loops connecting sTM 1 and sTM 2 as well as sTM 3 and sTM 4 are located extracellularly. Based on the lack of FLAG-antibody binding we suggest intracellular locations for the NH2- and COOH-termini and the region connecting sTM 4 and sTM 5. Immunoprecipitation studies of in vitro translated protein also suggest that the NH2-terminus is sited extracellularly. In immunohistochemical studies with NaPi-2-transfected MDCK cells, an interaction with NH2- and COOH- terminal antipeptide antibodies could only be obtained after membrane permeabilization. The presented data are an experimental documentation of the intracellular location of the NH2- and COOH-termini, and of the extracellular location of extracellular loops 1 and 2.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/185731
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