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

Pernille Bøttger,Lene Pedersen

doi:10.1186/1471-2091-12-21

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.

Highlights

The inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na+- or H +-dependent Pi-transporting proteins with representatives from all kingdoms
Human PiT1 E70 and human PiT2 H502 are critical for Pi transport function but dispensable for gamma-retroviral receptor function In a former study, we identified the putative 2nd-TM domain-positioned human PiT2 E55 as being critical for PiT2 Pi transport function (Figure 1) [28]
Besides being Pi-transporting proteins, the mammalian PiT proteins serve as gamma-retroviral receptors, and this dual-function allows for analyzing whether a mutated PiT protein is properly processed, folded and translocated to the cell surface [18,28]

Summary

Introduction

The inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na+- or H +-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian type III transporters are part of the Pi transport (PiT) family (SLC20 [5]; TC #2.A.20 [6]), but several members were originally identified as receptors for different retroviruses belonging to the gammaretrovirus genus [7,8,9,10,11,12,13]; PiT1 and PiT2 are proteins with dual functions. Gov/) that show similarity to the known members of the PiT family and are denoted putative phosphate permeases; and PiT family members have been found in all kingdoms [18], reviewed in [19] This suggests that the PiT proteins developed very early in evolution and that this family of proteins has important function(s) in all kingdoms of life

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