The Na+-phosphate cotransport system (NaPi-Π) with a cleaved protein backbone:: implications on function and membrane insertion

1. Renal handling or Inorganic phosphate (P-i) involves a Na+-P-i cotransport system which is well conserved between vertebrates. The members of this protein family, denoted NaPi-pi, share a topology with, it is thought, eight transmembrane domains. The transporter is proposed to be proteolytically cleaved within a large hydrophilic loop in vivo. 2. The consequences of an interrupted backbone were tested by constructing cDNA clones encoding different N-(1-3 and 1-5) and C-terminal (4-8 and 6-8) complementary fragments of NaPi-pi from winter flounder. When the cognate fragments were used in combination (1-3 plus 4-8; 1-5 plus 6-8) they comprised the full complement of the putative transporter domains. 3. None of the four individual fragments or the 1-5 plus 6-8 combination when expressed in Xenopus oocytes increased P-i flux. Coexpression of fragments 1-3 plus 4-8 stimulated transport activity identical to that for expressed wild-type NaPi-pi with regard to pH dependency and K-m for Na+ and P-i binding; however, the maximal transport rate (v(max)) was lower. 4. Immunohistochemistry on cryosections confined the functionally active 1-3 plus 4-8 combination to the oocyte membrane. This was not the case for the 1-5 plus 6-8 combination or any of the individual fragments, all of which failed to induce fluorescence. 5. A second immunohistochemical approach using intact oocytes allowed determination of the extracellular regions of the protein. Epitopes within the loop between transmembrane domains 3 and 4 enhanced fluorescence. Neither N- nor C-terminal tags induced fluorescence.