title: Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor: THE ROLE OF TYR179 IN FUNCTIONAL REGULATION.
abstract: Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pitransceptor in Saccharomyces cerevisiae Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi: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 PiA comparison of this conformation with the model for Pirelease in PiPT revealed that Tyr179in Pho84 (Tyr150in PiPT) is not part of the Pibinding site. This difference may be due to a lack of detailed information on the Pirelease 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. Tyr179then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr179in regulating Pirelease was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Piis a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pitransport cycle of a transceptor.
authors.affiliation: the Linnæus University Centre for Biomaterials Chemistry, Linnæus University, SE-391 82 Kalmar, Sweden.
authors.forename: Dieter R
authors.identifier: http://orcid.org/0000-0002-0381-251X
authors.initials: DR
authors.lastname: Samyn
authors.affiliation: the Linnæus University Centre for Biomaterials Chemistry, Linnæus University, SE-391 82 Kalmar, Sweden.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Van der Veken
authors.affiliation: the Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, BE-3001 Leuven-Heverlee, Flanders, Belgium, and.|the Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, BE-3001 Leuven-Heverlee, Flanders, Belgium.
authors.forename: Griet
authors.initials: G
authors.lastname: Van Zeebroeck
authors.affiliation: the Linnæus University Centre for Biomaterials Chemistry, Linnæus University, SE-391 82 Kalmar, Sweden.
authors.forename: Bengt L
authors.initials: BL
authors.lastname: Persson
authors.affiliation: the Linnæus University Centre for Biomaterials Chemistry, Linnæus University, SE-391 82 Kalmar, Sweden, bjorn.karlsson@lnu.se.|From the Computational Chemistry & Biochemistry Group.
authors.forename: Björn C G
authors.identifier: http://orcid.org/0000-0002-7392-0591
authors.initials: BC
authors.lastname: Karlsson
mesh_terms: D001693:Biological Transport, Active / Q000502:physiology; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D056004:Molecular Dynamics Simulation; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D028081:Proton-Phosphate Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C105495:PHO84 protein, S cerevisiae; D010710:Phosphates; D028081:Proton-Phosphate Symporters; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine
keywords: Pho84; Phosphate transport; Saccharomyces cerevisiae; Transceptor; cell signaling; membrane transport; molecular dynamics; yeast metabolism