Dataset Information

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 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.

SUBMITTER: Samyn DR

PROVIDER: S-EPMC5159500 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor: THE ROLE OF TYR179 IN FUNCTIONAL REGULATION.

Samyn Dieter R DR Van der Veken Jeroen J Van Zeebroeck Griet G Persson Bengt L BL Karlsson Björn C G BC

The Journal of biological chemistry 20161108 51

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 limi ...[more]

PMID: 27875295

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Project description:Protein folding is often considered the flux controlling process in protein synthesis and secretion. Here, two previously isolated Saccharomyces cerevisiae strains with increased α-amylase productivity were analyzed in chemostat cultures at different dilution rates using multi-omics data. Based on the analysis, we identified different routes of the protein folding pathway to improve protein production. In the first strain, the increased abundance of proteins working on the folding process, coordinated with upregulated glycogen metabolism and trehalose metabolism, helped increase α-amylase productivity 1.95-fold compared to the level in the original strain in chemostat culture at a dilution rate of 0.2/h. The second strain further strengthened the folding precision to improve protein production. More precise folding helps the cell improve protein production efficiency and reduce the expenditure of energy on the handling of misfolded proteins. As calculated using an enzyme-constrained genome-scale metabolic model, the second strain had an increased productivity of 2.36-fold with lower energy expenditure than that of the original under the same condition. Further study revealed that the regulation of N-glycans played an important role in the folding precision control and that overexpression of the glucosidase Cwh41p can significantly improve protein production, especially for the strains with improved folding capacity but lower folding precision. Our findings elucidated in detail the mechanisms in two strains having improved protein productivity and thereby provided novel insights for industrial recombinant protein production as well as demonstrating how multi-omics analysis can be used for identification of novel strain-engineering targets.IMPORTANCE Protein folding plays an important role in protein maturation and secretion. In recombinant protein production, many studies have focused on the folding pathway to improve productivity. Here, we identified two different routes for improving protein production by yeast. We found that improving folding precision is a better strategy. Dysfunction of this process is also associated with several aberrant protein-associated human diseases. Here, our findings about the role of glucosidase Cwh41p in the precision control system and the characterization of the strain with a more precise folding process could contribute to the development of novel therapeutic strategies.

Dataset Information

Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor: THE ROLE OF TYR179 IN FUNCTIONAL REGULATION.

Publications

Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor: THE ROLE OF TYR179 IN FUNCTIONAL REGULATION.

Similar Datasets

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