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)
Highlights
Inorganic phosphate (Pi) is an essential nutrient for almost all organisms, and is indispensable for the biosynthesis of cellular components such as nucleic acids, nucleoproteins, and phospholipids [1]
It has been shown that Pho89 mediates the cotransport of one inorganic phosphate (Pi) with two Na+ per transport cycle, and that its transport activity is driven by the electrical gradient (Dw) across the plasma membrane [11]
P. pastoris appears to be an attractive host for recombinant protein expression, because of: (a) the ability to grow to high cell density in defined media and the ease of scaling up; (b) the presence of the very strong and tightly regulated methanol-inducible alcohol oxidase 1 promoter; and (c) the ability to perform the eukaryotic post-translational modifications and integrate the expression plasmids in its own genome in one or more specific sites by homologous recombination [24,25]
Summary
Inorganic phosphate (Pi) is an essential nutrient for almost all organisms, and is indispensable for the biosynthesis of cellular components such as nucleic acids, nucleoproteins, and phospholipids [1]. Pho functions as a high-affinity cation-dependent Pi cotransporter that plays an obligatory role in the regulation of Pi homeostasis under alkaline growth conditions [8]. It shows optimal functional activity at pH 9.5, with a Km for phosphate of 0.5 lM [9]. Among the PiT family members, hPit and hPit have been well characterized at the cellular level by use of heterologous cell expression systems (fibroblast cells of murine origin and Xenopus laevis oocytes) [19,20]. The biochemical and biophysical characterization of the PiT family members is very limited, probably because of difficulties in obtaining substantial amounts of homogeneous functional protein. This study represents the first report on the functional reconstitution of a Pi-coupled PiT family member
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