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

Pflugers Arch. 1999 May;437(6):972-8. doi: 10.1007/s004240050869.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Carrier Proteins / chemistry*
  • Cell Line
  • Dogs
  • Epitopes / chemistry
  • Female
  • Glycosylation
  • Immunohistochemistry
  • Immunosorbent Techniques
  • Kidney Tubules, Proximal / chemistry*
  • Kidney Tubules, Proximal / ultrastructure
  • Microvilli / chemistry
  • Molecular Sequence Data
  • Oligopeptides
  • Oocytes
  • Peptide Fragments / chemistry
  • Peptides / chemistry
  • Peptides / immunology
  • Rats
  • Sodium-Phosphate Cotransporter Proteins
  • Sodium-Phosphate Cotransporter Proteins, Type II
  • Symporters*
  • Transfection
  • Xenopus laevis

Substances

  • Carrier Proteins
  • Epitopes
  • Oligopeptides
  • Peptide Fragments
  • Peptides
  • Sodium-Phosphate Cotransporter Proteins
  • Sodium-Phosphate Cotransporter Proteins, Type II
  • Symporters
  • FLAG peptide