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

Olsson, Anne LU (2000)
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... (More)
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. (Less)
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author
opponent
  • Prof Aducci, Patrizia, Dept. of Biology, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Roma, Italien
organization
publishing date
type
Thesis
publication status
published
subject
keywords
phosphorylation, fusicoccin, 14-3-3, C-terminus, autoinhibitory domain, plasma membrane, plant, H+-ATPase, Plant biochemistry, Växtbiokemi
pages
106 pages
publisher
Maivi Åkesson, växtbiokemi, Lunds Universitet
defense location
Sölvegatan 35, Lund
defense date
2000-06-09 10:15
external identifiers
  • Other:ISRN: LUNKDL/NK VK-00/1016
ISBN
91-973252-5-2
language
English
LU publication?
yes
id
5a669bb1-7afb-4d43-97d8-fe6f88395e0e (old id 40606)
date added to LUP
2007-10-14 16:58:43
date last changed
2016-09-19 08:45:15
@misc{5a669bb1-7afb-4d43-97d8-fe6f88395e0e,
  abstract     = {The plant plasma membrane H&lt;sup&gt;+&lt;/sup&gt;-ATPase is a predominant membrane enzyme that provides the energy for secondary active transport across the plasma membrane. Consequently, the H&lt;sup&gt;+&lt;/sup&gt;-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<br/><br>
<br/><br>
The plant plasma membrane H&lt;sup&gt;+&lt;/sup&gt;-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&lt;sub&gt;m&lt;/sub&gt;, a higher V&lt;sub&gt;max&lt;/sub&gt;, a more alkaline pH optimum, and an improved coupling between ATP hydrolysis and proton pumping.<br/><br>
<br/><br>
Characterization of the H&lt;sup&gt;+&lt;/sup&gt;-ATPase activities of leaf and root plasma membranes from tobacco (&lt;i&gt;Nicotiana tabacum&lt;/i&gt;) 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&lt;sub&gt;max&lt;/sub&gt;, and another regulating K&lt;sub&gt;m&lt;/sub&gt; and pH optimum.<br/><br>
<br/><br>
The fungal toxin fusicoccin activates the H&lt;sup&gt;+&lt;/sup&gt;-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&lt;sup&gt;+&lt;/sup&gt;-ATPase and that fusicoccin stabilizes a 14-3-3/H&lt;sup&gt;+&lt;/sup&gt;-ATPase complex, which represents the activated state of the enzyme.<br/><br>
<br/><br>
14-3-3 proteins bind to phosphorylated motifs in their target proteins. &lt;i&gt;In vivo&lt;/i&gt; phosphorylation of the plasma membrane H&lt;sup&gt;+&lt;/sup&gt;-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&lt;sub&gt;948&lt;/sub&gt;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&lt;sub&gt;948&lt;/sub&gt;V. Finally, we demonstrate that the phosphothreonine motif is important for the binding of 14-3-3, and hence in the activation the H&lt;sup&gt;+&lt;/sup&gt;-ATPase, also &lt;i&gt;in vivo&lt;/i&gt;.<br/><br>
<br/><br>
Taken together our data show that 14-3-3 is a natural ligand of the H&lt;sup&gt;+&lt;/sup&gt;-ATPase regulating H&lt;sup&gt;+&lt;/sup&gt;-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.},
  author       = {Olsson, Anne},
  isbn         = {91-973252-5-2},
  keyword      = {phosphorylation,fusicoccin,14-3-3,C-terminus,autoinhibitory domain,plasma membrane,plant,H+-ATPase,Plant biochemistry,Växtbiokemi},
  language     = {eng},
  pages        = {106},
  publisher    = {ARRAY(0x9fc8f78)},
  title        = {The Plant Plasma Memrbane H<sup>+</sup>-ATPase: regulation by phosphorylation and 14-3-3 proteins},
  year         = {2000},
}