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-ter minus of NaPi-2, was purified from renal brush-border membranes using non-r educing and then reducing column electrophoresis followed by enzymatic degl ycosylation and SDS-PAGE. The N-terminal sequence obtained for this fragmen t, VEAIG, indicates that the formation of p45 and p40 arises from the cleav age of p70 between arginine-319 and valine-320, In order to determine the m embrane topography of NaPi-2, brush-border membrane vesicles were digested with various proteases and the transporter-derived proteolytic peptides wer e subsequently identified by Western blotting using N- and C-terminal-direc ted antibodies. Our results lead us to propose an alternative topographical model in which p45 and p40 possess three transmembrane domains each and in dicate 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 lo calization of the cleavage site indicated that NaPi-2 could either be proce ssed intracellularly by vesicular proteases or extracellularly by secretory proteases or by brush-border membrane ectoenzymes. (C) 1999 Published by E lsevier Science B.V. All rights reserved.