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Key Residues and Phosphate Release Routes in the Saccharomyces cerevisae Pho84 Transceptor - The Role of Tyr179 in Functional Regulation
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Cocordia University, Canada. (BMC)ORCID iD: 0000-0002-0381-251X
Inst Agr & Fisheries Res ILVO, Belgium.
VIB, Belgium ; Katholieke Univ Leuven, Belgium.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. (BMC)
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2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 51, p. 26388-26398Article in journal (Refereed) Published
Abstract [en]

Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor 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 Pi. A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr(179) in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr(179), Asp(178), is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr(179) then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr(179) in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.

Place, publisher, year, edition, pages
2016. Vol. 291, no 51, p. 26388-26398
Keyword [en]
Pho84, Saccharomyces cerevisiae, Molecular dynamics, membrane protein, transport
National Category
Biochemistry and Molecular Biology Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
URN: urn:nbn:se:lnu:diva-60171DOI: 10.1074/jbc.M116.738112ISI: 000391568200013Scopus ID: 2-s2.0-85006241160OAI: oai:DiVA.org:lnu-60171DiVA, id: diva2:1068227
Funder
The Crafoord Foundation, 20150874Swedish National Infrastructure for Computing (SNIC), SNIC 2014/1-404 SNIC 2015/1-444
Available from: 2017-01-24 Created: 2017-01-24 Last updated: 2017-06-27Bibliographically approved

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