Functional expression, purification and reconstitution of the recombinant phosphate transporter Pho89 of Saccharomyces cerevisiae

Abstract

The Saccharomyces cerevisiae high-affinity phosphate transporter Pho89 is a member of the inorganic phosphate (Pi) transporter (PiT) family, and shares significant homology with the type III Na+/Pi symporters, hPit1 and hPit2. Currently, detailed biochemical and biophysical analyses of Pho89 to better understand its transport mechanisms are limited, owing to the lack of purified Pho89 in an active form. In the present study, we expressed functional Pho89 in the cell membrane of Pichia pastoris, solubilized it in Triton X-100 and foscholine-12, and purified it by immobilized nickel affinity chromatography combined with size exclusion chromatography. The protein eluted as an oligomer on the gel filtration column, and SDS/PAGE followed by western blotting analysis revealed that the protein appeared as bands of approximately 63, 140 and 520 kDa, corresponding to the monomeric, dimeric and oligomeric masses of the protein, respectively. Proteoliposomes containing purified and reconstituted Pho89 showed Na+-dependent Pi transport activity driven by an artificially imposed electrochemical Na+ gradient. This implies that Pho89 operates as a symporter. Moreover, its activity is sensitive to the Na+ ionophore monensin. To our knowledge, this study represents the first report on the functional reconstitution of a Pi-coupled PiT family member.Structured digital abstractPho89 and Pho89 bind by molecular sieving (View interaction)Pho89 and Pho89 bind by comigration in gel electrophoresis (View interaction)Pho89 and Pho89 bind by molecular sieving (View interaction)Pho89 and Pho89 bind by comigration in gel electrophoresis (View interaction)

Structure of the sodium-dependent phosphate transporter reveals insights into human solute carrier SLC20

“…PiTs are present in all kingdoms, including sodium-dependent PiT from Saccharomyces cerevisiae Sc PHO89 ( 12 ), liverwort Marchantia polymorpha Mp PTB ( 13 ), and Plasmodium falciparum Pf PiT ( 14 ) or proton-dependent PiT from Arabidopsis thaliana At Pht2 ( 15 ) and Escherichia coli Ec PitA ( 16 ). These PiT proteins have a transmembrane region at both termini linked by a loop or an additional intracellular soluble domain (S domain) in the cellular region (>200 residues) (fig.…”

A Functional Approach towards Understanding the Role of the Mitochondrial Respiratory Chain in an Endomycorrhizal Symbiosis

“…Within the putative low affinity P transporters, grouping with the yeast transporters ScPHO87 and ScPHO90 described by Pinson et al (2004), only one R. irregulare gene was found ( RiPT7 ). The third cluster groups sequences presented homology with the S. cerevisiae high affinity Na + /Pi cotransporter ScPHO89 (Sengottaiyan et al, 2013) and two R. irregulare sequences ( RiPT5 and RiPT6 ). RiPT6 presented an incomplete sequence and no expression in our experimental conditions.…”

Co‐metabolism of Na+/K+ ion regulated physiological enhancement on selenium‐accumulation in Saccharomyces yeasts

“…Finally, the cells should find it hard to transport K + through the sodium‐potassium pump due to the high energy barrier in the Na 2 SeO 3 group. Furthermore, under low phosphate content, cell growth shall be highly blocked without sufficient K + maintenance in physiological activities 40 …”

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