Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium‐dependent phosphate transport function of human PiT2

The mammalian members of the inorganic phosphate (Pi) transporter (PiT) family, the type III sodium‐dependent phosphate (NaPi) transporters PiT1 and PiT2, have been assigned housekeeping Pi transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na+) or proton (H+) gradients to transport Pi. Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N‐ and C‐terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N‐ and C‐terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed Pi uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaPi transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild‐type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na+‐dependent Pi transport function of human PiT2. The conservation of the aspartates among proteins using either Na+‐ or H+‐gradients for Pi transport suggests that they are involved in H+‐dependent Pi transport as well. Current results favor a membrane topology model in which the N‐ and C‐terminal PiT family signature sequences are positioned in intra‐ and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.