Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity P_i transceptor in Saccharomyces cerevisiae Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by P_i:H⁺ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of P_i A comparison of this conformation with the model for P_i release in PiPT revealed that Tyr¹⁷⁹ in Pho84 (Tyr¹⁵⁰ in PiPT) is not part of the P_i binding site. This difference may be due to a lack of detailed information on the P_i release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr¹⁷⁹, Asp¹⁷⁸, is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr¹⁷⁹ then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr¹⁷⁹ in regulating P_i release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of P_i is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled P_i transport cycle of a transceptor.

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Journal Article

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Samyn DR, Van der Veken J, Van Zeebroeck G, Persson BL, Karlsson BC

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