Studies on the topology of the renal type II NaPi-cotransporter.
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.
Lambert
G
G
Physiologisches Institut, Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Traebert
M
M
Hernando
N
N
Biber
J
J
Murer
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D002460:Cell Line; D004285:Dogs; D000939:Epitopes / Q000737:chemistry; D005260:Female; D006031:Glycosylation; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D007687:Kidney Tubules, Proximal / Q000737:chemistry* / Q000648:ultrastructure; D008871:Microvilli / Q000737:chemistry; D008969:Molecular Sequence Data; D009842:Oligopeptides; D009865:Oocytes; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D051381:Rats; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters; D014162:Transfection; D014982:Xenopus laevis
D002352:Carrier Proteins; D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters; C085706:FLAG peptide