Functional expression of rat renal Na/Pi-cotransport (NaPi-2) in Sf9 cells by the baculovirus system.
The recently cloned Na/Pi-cotransport system NaPi-2 is an apical membrane protein of rat proximal tubular cells and is involved in proximal phosphate reabsorption. To make the protein available for further functional/structural studies, this transport system has been expressed in Sf9 insect cells using a recombinant baculovirus. Sf9 cells infected with NaPi-2 (or 6His tagged NaPi-2) expressed functional Na/Pi-cotransport up to 20- to 50-fold over noninfected Sf9 cells. Transport of phosphate in infected cells was highly dependent on sodium, exhibited a Km for Pi of 0.114 mM and an apparent Km for Na of 63 mM (Hill coefficient of approximately 3) and was stimulated by high external pH. Infected cells expressed a polypeptide of 65 kDa representing a nonglycosylated form of the 85 kDa mature NaPi-2 transporter as present in proximal tubular brush-border membranes. By confocal microscopy expression of NaPi-2 protein was observed in the plasma membrane, yet submembranous accumulation of NaPi-2 protein could not be excluded. This demonstrates that the rat proximal tubular Na/Pi-cotransport system NaPi-2 can be successfully expressed in Sf9 cells with characteristics similar to that in isolated brush-border membranes. The 6His tagging will permit isolation of the NaPi-2 cotransporter in amounts sufficient for structural/functional studies.
Fucentese
M
M
Laboratory of Biochemistry I, ETH-Zürich, Switzerland.
Winterhalter
K
K
Murer
H
H
Biber
J
J
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D007668:Kidney / Q000737:chemistry*; D018613:Microscopy, Confocal; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*; D027981:Symporters
D002352:Carrier Proteins; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D012964:Sodium