Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2.
The general phosphate need in mammalian cells is accommodated by members of the P(i) transport (PiT) family (SLC20), which use either Na(+) or H(+) to mediate inorganic phosphate (P(i)) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na(+)-dependent P(i) (NaP(i)) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with (32)P(i) as a traceable P(i) source. For PiT1, the Michaelis-Menten constant for P(i) was determined as 322.5 +/- 124.5 microM. PiT2 was analyzed for the first time and showed positive cooperativity in P(i) uptake with a half-maximal activity constant for P(i) of 163.5 +/- 39.8 microM. PiT1- and PiT2-mediated Na(+)-dependent P(i) uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na(+) dependency patterns. However, only PiT2 was capable of Na(+)-independent P(i) transport at acidic pH. Study of the impact of divalent cations Ca(2+) and Mg(2+) revealed that Ca(2+) was important, but not critical, for NaP(i) transport function of PiT proteins. To gain insight into the NaP(i) cotransport function, we analyzed PiT2 and a PiT2 P(i) transport knockout mutant using (22)Na(+) as a traceable Na(+) source. Na(+) was transported by PiT2 even without P(i) in the uptake medium and also when P(i) transport function was knocked out. This is the first time decoupling of P(i) from Na(+) transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E(55) and E(575) are responsible for linking P(i) import to Na(+) transport in PiT2.
Bøttger
Pernille
P
Department of Molecular Biology, Institute of Clinical Medicine, University of Aarhus, Aarhus, Denmark.
Hede
Susanne E
SE
Grunnet
Morten
M
Høyer
Boy
B
Klaerke
Dan A
DA
Pedersen
Lene
L
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D018075:RNA, Complementary / Q000378:metabolism; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D012979:Sodium Radioisotopes / Q000378:metabolism; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D020033:Protein Isoforms; D018075:RNA, Complementary; D012979:Sodium Radioisotopes; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D012964:Sodium; D004262:DNA Restriction Enzymes; D008274:Magnesium; D002118:Calcium