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Regulation of H+-pyrophosphatase by 14-3-3 Proteins from Arabidopsis thaliana

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Abstract

Plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a crucial enzyme that exists on the tonoplast to maintain pH homeostasis across the vacuolar membrane. This enzyme generates proton gradient between cytosol and vacuolar lumen by hydrolysis of a metabolic byproduct, pyrophosphate (PP i ). The regulation of V-PPase at protein level has drawn attentions of many workers for decades, but its mechanism is still unclear. In this work, we show that AVP1, the V-PPase from Arabidopsis thaliana, is a target protein for regulatory 14-3-3 proteins at the vacuolar membrane, and all twelve 14-3-3 isoforms were analyzed for their association with AVP1. In the presence of 14-3-3ν, -µ, -ο, and -ι, both enzymatic activities and its associated proton pumping of AVP1 were increased. Among these 14-3-3 proteins, 14-3-3 µ shows the highest stimulation on coupling efficiency. Furthermore, 14-3-3ν, -µ, -ο, and -ι exerted protection of AVP1 against the inhibition of suicidal substrate PP i at high concentration. Moreover, the thermal profile revealed the presence of 14-3-3ο improves the structural stability of AVP1 against high temperature deterioration. Additionally, the 14-3-3 proteins mitigate the inhibition of Na+ to AVP1. Besides, the binding sites/motifs of AVP1 were identified for each 14-3-3 protein. Taken together, a working model was proposed to elucidate the association of 14-3-3 proteins with AVP1 for stimulation of its enzymatic activity.

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Abbreviations

V-PPase:

Vacuolar H+-pyrophosphate

V-ATPase:

Vacuolar H+-ATPase

TMs:

Transmembrane helices

DDM:

n-Dodecyl β-d-maltoside

Ni2+–NTA:

Nickel–nitrilotriacetic acid

PMSF:

Phenylmethylsulfonyl fluoride

MYTH:

Split-ubiquitin membrane yeast two-hybrid

3-AT:

3-Aminotriazole

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Acknowledgements

This work was supported by Grants from Ministry of Science and Technology, Republic of China to R.-L. Pan (NSC 103-2311-B-007-001-MY2, NSC 104-2321-B-007-002, NSC 105-2311-B-007-011-MY2), and by the Grants of the thematic research program from Agricultural Biotechnology Research Center, Academia Sinica, Republic of China (AS 103-TP-B11).

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  1. Yu-Di Hsu and Yu-Fen Huang have contributed equally to this work.

Authors and Affiliations

  1. Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University, Hsin Chu, 30013, Taiwan, Republic of China

    Yu-Di Hsu, Yu-Fen Huang, Yih-Jiuan Pan, Li-Kun Huang, Ya-Yun Liao, Wei-Hua Lin, Tzu-Yin Liu, Ching-Hung Lee & Rong-Long Pan

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  1. Yu-Di Hsu
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Contributions

YDH and YFH performed the experiments. All authors have participated in the discussions and technique assistance during the course of this studies. YDH wrote the paper initially. CHL and RLP supervised the experimental design of this project and approved the final version of the manuscript.

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Correspondence to Ching-Hung Lee or Rong-Long Pan.

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Hsu, YD., Huang, YF., Pan, YJ. et al. Regulation of H+-pyrophosphatase by 14-3-3 Proteins from Arabidopsis thaliana. J Membrane Biol 251, 263–276 (2018). https://doi.org/10.1007/s00232-018-0020-4

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