Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor

Abstract

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 Tyr179 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 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 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.

Structure–Function Analysis Reveals Amino Acid Residues of Arabidopsis Phosphate Transporter AtPHT1;1 Crucial for Its Activity

“…We next measured the 33 Pi uptake activities of pam2 -vector and pam2 - At PHT1;1 grown under a low Pi concentration (100 μM KH 2 PO 4 ). A similar Pi concentration was used in previous studies on Sc PHO84 (Samyn et al, 2012; Samyn et al, 2016). To examine the pH dependence, we determined the transport activity of pam2 - At PHT1;1 in the solution with different pH (pH 4.0 to 7.0) for 20 min and found its activity increased concomitantly with the decrease of pH ( Figure 3C ), suggesting the coupling of H + with Pi transport.…”

“…Based on the structure model of Pi PT proposed previously, D35 (TM1), D38 (TM1), R134 (TM4), and D144 (TM4) are suggested to interact with H + . The rest, Y145 (TM4), F169 (TM5), Q172 (TM5), W304 (TM7), D308 (TM7), Y312 (TM7), N421 (TM10), and K449 (TM11), are believed to reside in the putative Pi binding pocket ( Figure 2 and Table S2 ; Samyn et al, 2012; Pedersen et al, 2013; Samyn et al, 2016). It is also believed that the residues in core helices of TMs 1, 4, 7, and 10 at the central path of MFS members possibly engage in substrate translocation, while those in the middle helices of TMs 2, 5, 8, and 11 participate in substrate binding and co-transport coupling (Yan, 2015).…”

“…Additionally, the aromatic moieties of the Y145 and F169 residues ( Figure 1B ) and the electrostatic interactions derived from the hydrogen bonds within the residues of Y312-Q172-Y359-N417-T355, W304-D308, and K449-T81 located in the Pi binding pocket ( Figures S2A, B, H, I, K ) could stabilize the Pi binding. The conserved residue of D358 in Sc PHO84 and D324 in Pi PT (corresponding to D308 in At PHT1;1) were suggested to participate directly in Pi transport by protonation/deprotonation to trigger off conformational changes ( Table S2 ; Samyn et al, 2012; Pedersen et al, 2013; Samyn et al, 2016). In our analysis, however, the D308A variant still retained partial activity ( Figure 4B ) and the D308 residue could form electrostatic interaction with the W304 residue ( Figure S2H ).…”

“…The transport mechanism of Pi PT was proposed to be mediated by conformational changes through protonation and deprotonation of D324 residue, triggering off an outward open state to a ligand-bound occluded state (Pedersen et al, 2013). The functional analysis of Sc PHO84 suggested the involvement of R168 and D178 residues (corresponding to R139 and D149 residues in Pi PT) in H + transfer and Y179, D358, and K492 residues (corresponding to Y150, D324, and K459 residues in Pi PT) in the Pi-binding pocket (Samyn et al, 2012; Samyn et al, 2016). Nevertheless, the importance of these amino acid residues is not validated experimentally in vivo .…”

Multiple Transceptors for Macro- and Micro-Nutrients Control Diverse Cellular Properties Through the PKA Pathway in Yeast: A Paradigm for the Rapidly Expanding World of Eukaryotic Nutrient Transceptors Up to Those in Human Cells

“…Gly3P is transported into the cells by the Git1 and Pho91 carriers, but in this case there is no signaling to PKA ( Popova et al, 2010 ). Second, by insertion of specific point mutations in the putative H + -binding residues ( Samyn et al, 2012 , 2016 ). This abolishes the transport of phosphate through the Pho84 transporter, but apparently still allows proper binding of phosphate into the substrate-binding pocket as well as proper induction of the conformational change that triggers signaling to the PKA pathway.…”