Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2.
The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular structure of NaPi-2.
Xiao
Y
Y
Laboratoire d'oncologie moléculaire, Département de chimie-biochimie, Université du Québec à Montréal, P.O. Box 8888, Centre-Ville Station, Montreal, Que., H3C 3P8, Canada.
Boyer
C J
CJ
Vincent
E
E
Dugré
A
A
Vachon
V
V
Potier
M
M
Béliveau
R
R
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007668:Kidney / Q000737:chemistry*; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010720:Phosphines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D002352:Carrier Proteins; D004220:Disulfides; D004229:Dithiothreitol; D007202:Indicators and Reagents; D008623:Mercaptoethanol; D010710:Phosphates; D010720:Phosphines; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; C080938:tris(2-carboxyethyl)phosphine