Membrane topography of the renal phosphate carrier NaPi-2: limited proteolysis studies.
The rat sodium/phosphate cotransporter NaPi-2 is a 70 kDa polypeptide (p70) for which eight transmembrane segments have been predicted. We have shown that p70 exists predominantly as p45 and p40 fragments which are linked by disulfide bonds. In this work, the p40 fragment, corresponding to the C-terminus of NaPi-2, was purified from renal brush-border membranes using non-reducing and then reducing column electrophoresis followed by enzymatic deglycosylation and SDS-PAGE. The N-terminal sequence obtained for this fragment, VEAIG, indicates that the formation of p45 and p40 arises from the cleavage of p70 between arginine-319 and valine-320. In order to determine the membrane topography of NaPi-2, brush-border membrane vesicles were digested with various proteases and the transporter-derived proteolytic peptides were subsequently identified by Western blotting using N- and C-terminal-directed antibodies. Our results lead us to propose an alternative topographical model in which p45 and p40 possess three transmembrane domains each and indicate that the processing site of p70 for the generation of p45 and p40 is localized in a large protein core facing the extracellular milieu. This localization of the cleavage site indicated that NaPi-2 could either be processed intracellularly by vesicular proteases or extracellularly by secretory proteases or by brush-border membrane ectoenzymes.
Paquin
J
J
Laboratoire d'oncologie moléculaire, Département de chimie-biochimie et Hôpital Ste-Justine, Université du Québec à Montréal, P.O. Box 8888, Centre-Ville Station, Montreal, Que. H3C 3P8, Canada.
Vincent
E
E
Dugré
A
A
Xiao
Y
Y
Boyer
C J
CJ
Béliveau
R
R
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D007668:Kidney / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008871:Microvilli / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D014357:Trypsin
D002352:Carrier Proteins; D010446:Peptide Fragments; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D002918:Chymotrypsin; D014357:Trypsin; D019286:Endopeptidase K