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

Abstract

The mammalian members of the inorganic phosphate (P i ) transporter (PiT) family, the type III sodium-dependent phosphate (NaP i ) transporters PiT1 and PiT2, have been assigned housekeeping P i 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 P i . 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 P i uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaP i 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 P i transport function of human PiT2. The conservation of the aspartates among proteins using either Na + -or H + -gradients for P i transport suggests that they are involved in H + -dependent P i 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.Abbreviations 10A1, an MLV isolate related to AM-MLV; AM-MLV, amphotropic MLV; CHO, Chinese hamster ovary; FeLV-B, feline leukemia virus subgroup B; GALV, gibbon ape leukemia virus; MLV, murine leukemia virus; NaP i , sodium-dependent phosphate; PiT, inorganic phosphate transporter; RADAR, rapid automatic detection and alignment of repeats.

Molecular Mechanisms of Phosphate Homeostasis in <i>Escherichia coli</i>

“…This signature sequence is highlighted with red letters in Figure 1A. The human proteins from this family are thought to be involved in housekeeping functions and are called PiT1 and PiT2, whereas the Neurospora crassa and Saccharomyces cerevisiae members are called Pho-4 and Pho89, respectively [32,33]. Mutations in the signature sequence of the PiT2 protein block Pi transport [34].…”

“…Mutations in the signature sequence of the PiT2 protein block Pi transport [34]. In addition to their role in Pi transport, the PiT1 and PiT2 proteins are also receptors for the gamma-retroviruses [32]. This protein family includes both Na + -dependent and H + -dependent Pi symporters.…”

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

“…Nevertheless, the analyses of a P i transport knockout PiT2 mutant protein, PiT2 E 55 Q E 91 Q E 575 Q, did provide knowledge on the mechanism of the Na ϩ transport, showing that it can occur even though the P i uptake function is abolished. The glutamates in human PiT2 positions 55, 91, and 575 are believed to locate to transmembrane domains (4,9,14,32). A former study (5) revealed that E 91 is dispensable, whereas E 55 and E 575 are critical for P i transport of PiT2.…”

“…Hydropathy profiles show that the PiT paralogs are polytopic proteins and predict 10 -12 transmembrane domains (4,9,14,32) and a large hydrophilic domain near the center of each protein that has been experimentally assigned to the cytoplasmic space in PiT2 (6).…”

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake

“…To this aim, we constructed hPiT1 and hPiT2 chimeric proteins expressing the eYFP acceptor or Rluc donor. Since structure-function studies have excluded a role of the large intracellular loop (iLoop) in Pi transport and retrovirus binding (59)(60)(61)(62), and showed no overlapping between iLoop and the highly hydrophobic domain (57), we substituted the iLoop with eYFP and Rluc sequences ( Figure 3C). When expressed in HEK293T, the chimeric hPiT1-eYFP or -Rluc and hPiT2-eYFP or -Rluc proteins could be visualized at the plasma membrane, as shown by confocal microscopy ( Figure 3D), enabling to study their role in detecting the variation of extracellular Pi levels.…”