Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life

Abstract

BackgroundThe inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na+- or H+-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with Pi to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na+-dependent Pi (NaPi) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins.ResultsWe show that the human PiT2 histidine, H502, and the human PiT1 glutamate, E70, - both conserved in eukaryotic PiT family members - are critical for Pi transport function. Noticeably, human PiT2 H502 is located in the C-terminal PiT family signature sequence, and human PiT1 E70 is located in ProDom domains characteristic for all PiT family members.A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR254-V483), was found to be a fully functional Pi transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL183-V483) did also support Pi transport albeit at very low levels.ConclusionsThe results suggest that the overall structure of the Pi-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship.

Deconstructing Fahr's disease/syndrome of brain calcification in the era of new genes

“…Although calcification is limited to brain in cases reported with SLC20A2, its role of in homeostasis for inorganic phosphate is evident in several other tissues around the body including bone, parathyroid, and kidneys [19,20].…”

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

“…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]. In addition to their role in Pi transport, the PiT1 and PiT2 proteins are also receptors for the gamma-retroviruses [32].…”

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.…”