The Plant Plasma Memrbane H+-ATPase: regulation by phosphorylation and 14-3-3 proteins

Anne Olsson

Research output: ThesisDoctoral Thesis (compilation)

Abstract

The plant plasma membrane H<sup>+</sup>-ATPase is a predominant membrane enzyme that provides the energy for secondary active transport across the plasma membrane. Consequently, the H<sup>+</sup>-ATPase is thought to play a major role in many cell processes, and it is implicated to be regulated by a number of factors, including hormones, blue light, and fungal toxins

The plant plasma membrane H<sup>+</sup>-ATPase is regulated via an autoinhibitory domain located within the C-terminal region of the enzyme. Removal or displacement of this regulatory domain results in an activated enzyme with a lower K<sub>m</sub>, a higher V<sub>max</sub>, a more alkaline pH optimum, and an improved coupling between ATP hydrolysis and proton pumping.

Characterization of the H<sup>+</sup>-ATPase activities of leaf and root plasma membranes from tobacco (<i>Nicotiana tabacum</i>) revealed a difference in the activation state of the enzymes in the two organs. This discovery led to the suggestion that there are at least two regulatory sites within the C-terminal autoinhibitory domain, one regulating V<sub>max</sub>, and another regulating K<sub>m</sub> and pH optimum.

The fungal toxin fusicoccin activates the H<sup>+</sup>-ATPase by a mechanism involving a displacement of the C-terminal autoinhibitory domain. We have shown that the fusicoccin ¨receptor¨, a 14-3-3 protein, binds directly to the C-terminal region of the H<sup>+</sup>-ATPase and that fusicoccin stabilizes a 14-3-3/H<sup>+</sup>-ATPase complex, which represents the activated state of the enzyme.

14-3-3 proteins bind to phosphorylated motifs in their target proteins. <i>In vivo</i> phosphorylation of the plasma membrane H<sup>+</sup>-ATPase from spinach leaves in the presence of fusicoccin made it possible to identify a phosphorylated amino acid in the C terminus. The phosphorylation of this amino acid residue, Thr-948, the penultimate amino acid in the C terminus, was protected by the fusicoccin-dependent binding of 14-3-3 to the C terminus. Characterization of this novel 14-3-3 binding motif, QQXYpT<sub>948</sub>V, revealed that phosphorylation of Thr-948 is a prerequisite for binding of 14-3-3. Moreover, we could show that the fusicoccin-dependent 14-3-3 binding occurs independently of phosphorylation but still involves the three ultimate amino acids, YT<sub>948</sub>V. Finally, we demonstrate that the phosphothreonine motif is important for the binding of 14-3-3, and hence in the activation the H<sup>+</sup>-ATPase, also <i>in vivo</i>.

Taken together our data show that 14-3-3 is a natural ligand of the H<sup>+</sup>-ATPase regulating H<sup>+</sup>-pumping, that phosphorylation of Thr-948 is a prerequisite for 14-3-3 binding, and that fusicoccin replaces the need for phosphorylation of Thr-948.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Biochemistry and Structural Biology
Supervisors/Advisors
  • [unknown], [unknown], Supervisor, External person
Award date2000 Jun 9
Publisher
Print ISBNs91-973252-5-2
Publication statusPublished - 2000

Bibliographical note

Defence details

Date: 2000-06-09
Time: 10:15
Place: Sölvegatan 35, Lund

External reviewer(s)

Name: Aducci, Patrizia
Title: Prof
Affiliation: Dept. of Biology, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Roma, Italien

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Article: Olsson, A., Johansson, F., Sommarin, M., and Larsson, C. (1995)Multiple regulatory sites in the C-terminal autoinhibitory domain of the plasma membrane H+-ATPase. Plant J. 8, 959-962

Article: Jahn, T., Fuglsang, T.A., Olsson, A., Brüntrup, I.M., Collinge, D.B., Volkmann, D., Sommarin, M., Palmgren, M.G., and Larsson, C. (1997)The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H+-ATPase. Plant Cell 9, 1805-1814

Article: Olsson, A., Svennelid, F., Ek, B., Sommarin, M., and Larsson, C. (1998)A phosphothreonine residue at the C-terminal end of the plasma membrane H+-ATPase is protected by fusicoccin-induced 14-3-3 binding. Plant Physiol. 118, 551-555

Article: Svennelid, F., Olsson, A., Piotrowski, M., Rosenquist, M., Ottman, C., Larsson, C., Oecking, C., and Sommarin, M. (1999)Phosphorylation of Thr-948 in the C terminus of the plasma membrane H+-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell 11, 2379-2392

Subject classification (UKÄ)

  • Biological Sciences

Keywords

  • phosphorylation
  • fusicoccin
  • 14-3-3
  • C-terminus
  • autoinhibitory domain
  • plasma membrane
  • plant
  • H+-ATPase
  • Plant biochemistry
  • Växtbiokemi

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