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The flip side of the Arabidopsis type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H⁺ gradient to synthesize pyrophosphate.

ABSTRACT: Energy partitioning and plant growth are mediated in part by a type I H⁺-pumping pyrophosphatase (H⁺-PPase). A canonical role for this transporter has been demonstrated at the tonoplast where it serves a job-sharing role with V-ATPase in vacuolar acidification. Here, we investigated whether the plant H⁺-PPase from Arabidopsis also functions in "reverse mode" to synthesize PP_i using the transmembrane H⁺ gradient. Using patch-clamp recordings on Arabidopsis vacuoles, we observed inward currents upon P_i application on the cytosolic side. These currents were strongly reduced in vacuoles from two independent H⁺-PPase mutant lines (vhp1-1 and fugu5-1) lacking the classical PP_i-induced outward currents related to H⁺ pumping, whereas they were significantly larger in vacuoles with engineered heightened expression of the H⁺-PPase. Current amplitudes related to reverse-mode H⁺ transport depended on the membrane potential, cytosolic P_i concentration, and magnitude of the pH gradient across the tonoplast. Of note, experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing the Arabidopsis H⁺-PPase (AVP1) demonstrated P_i-dependent PP_i synthase activity in the presence of a pH gradient. Our work establishes that a plant H⁺-PPase can operate as a PP_i synthase beyond its canonical role in vacuolar acidification and cytosolic PP_i scavenging. We propose that the PP_i synthase activity of H⁺-PPase contributes to a cascade of events that energize plant growth.

SUBMITTER: Scholz-Starke J

PROVIDER: S-EPMC6349097 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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The flip side of the Arabidopsis type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H+ gradient to synthesize pyrophosphate.

Scholz-Starke Joachim J Primo Cecilia C Yang Jian J Kandel Raju R Gaxiola Roberto A RA Hirschi Kendal D KD

The Journal of biological chemistry 20181203 4

Energy partitioning and plant growth are mediated in part by a type I H+-pumping pyrophosphatase (H+-PPase). A canonical role for this transporter has been demonstrated at the tonoplast where it serves a job-sharing role with V-ATPase in vacuolar acidification. Here, we investigated whether the plant H+-PPase from Arabidopsis also functions in "reverse mode" to synthesize PPi using the transmembrane H+ gradient. Using patch-clamp reco ...[more]

PMID: 30510138

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Project description:The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 ± 0.01 h-1) and the biomass yield by 38% (0.18 ± 0.00 gbiomass gsubstrate-1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk-malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PPi stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi, or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi- and ATP-dependent glycolysis.

Dataset Information

The flip side of the Arabidopsis type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H⁺ gradient to synthesize pyrophosphate.

Publications

The flip side of the <i>Arabidopsis</i> type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H<sup>+</sup> gradient to synthesize pyrophosphate.

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