The Na+-phosphate cotransport system (NaPi-II) with a cleaved protein backbone: implications on function and membrane insertion.
1. Renal handling of inorganic phosphate (Pi) involves a Na+-Pi cotransport system which is well conserved between vertebrates. The members of this protein family, denoted NaPi-II, 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-II 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 Pi flux. Coexpression of fragments 1-3 plus 4-8 stimulated transport activity identical to that for expressed wild-type NaPi-II with regard to pH dependency and Km for Na+ and Pi binding; however, the maximal transport rate (vmax) 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.
Kohl
B
B
Max-Planck-Institut fur molekulare Physiologie, Abteilung Epithelphysiologie, Rheinlanddamm 201, 44139 Dortmund, Germany.
Wagner
C A
CA
Huelseweh
B
B
Busch
A E
AE
Werner
A
A
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D000939:Epitopes; D005110:Extracellular Space / Q000378:metabolism; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008566:Membranes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D000939:Epitopes; D008565:Membrane Proteins; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D012313:RNA