The Plasma Membrane H+-ATPase - Identification of a 14-3-3 binding motif
Research output: Thesis › Doctoral Thesis (compilation)
The P-type plasma membrane H<sup>+</sup>-ATPases form a group of proteins only found in plants and fungi. The pumping of protons across the plasma membrane, energized by ATP hydrolysis, creates an electrochemical gradient that is essential for solute transport and internal pH regulation. The H<sup>+</sup>-ATPase genes are present as multigene families in the genomes of higher plants and all cell types investigated express some H<sup>+</sup>-ATPase gene. The fundamental importance of the electrochemical gradient makes precise regulation of the H<sup>+</sup>-ATPase important. Several internal and external factors, such as hormones, light, pH, and fungal toxins, are involved in regulating the plant plasma membrane H<sup>+</sup>-ATPase activity. Besides the direct regulation by pH and ATP availability, the activity is controlled by an autoinhibitory C-terminal domain in the H<sup>+</sup>-ATPase. Removing this C-terminal domain by proteolysis or by fusicoccin-induced 14-3-3 binding irreversibly activates the enzyme. Normally, 14-3-3 binds to phosphorylated motifs and by incubating spinach leaves with <sup>32</sup>P-orthophosphate and the fungal toxin fusicoccin <i>in vivo</i> it was possible to radiolabel the H<sup>+</sup>-ATPase. The radiolabeling could be removed by proteolysis and sequencing the released radiolabeled peptides identified the phosphorylated amino acid as the penultimate threonine in the C terminus, in the relatively conserved motif QQXYTV. This phosphorylated threonine is essential for 14-3-3 binding in the absence of fusicoccin, whereas fusicoccin-induced 14-3-3 binding occurs regardless of phosphorylation but still requires the YTV residues. The physiological importance of this motif was shown by heterologous expression of a plant H<sup>+</sup>-ATPase in yeast. Mutations in the motif abolished or heavily reduced 14-3-3 binding and activation of the plant H<sup>+</sup>-ATPase. <i>In vitro</i> phosphorylation of isolated plasma membranes with [ g -<sup>32</sup>P]ATP radiolabels the H<sup>+</sup>-ATPase in a calcium-dependent way and creates a 14-3-3 binding site in the H<sup>+</sup>-ATPase, containing a phosphothreonine. The H<sup>+</sup>-ATPase isoforms AHA1 and AHA2 are present in Arabidopsis leaf plasma membranes under normal conditions; fusicoccin treatment induces expression of three additional isoforms, AHA3, AHA8, and AHA11. Five 14-3-3 isoforms, epsilon, mu, nu, omega, and upsilon, are associated with the plasma membrane, where the H<sup>+</sup>-ATPase is the main target for 14-3-3 binding; after infiltration with fusicoccin there is a change in isoforms, omega disappears and the chi isoform appears. In summary, the data show that <i>in vivo</i> phosphorylation of the penultimate threonine in the motif QQXYTV regulates the H<sup>+</sup>-ATPase activity by 14-3-3 binding and that a calcium-dependent protein kinase activity phosphorylating this threonine <i>in vitro</i> is present in the plasma membrane. The appearance of additional H<sup>+</sup>-ATPase isoforms and the shift in 14-3-3 isoforms after fusicoccin-treatment is interesting and further research might answer questions regarding 14-3-3 isoform specificity and the function of the different H<sup>+</sup>-ATPase isoforms, in e.g. adaptation to stress.
|Research areas and keywords||
Subject classification (UKÄ) – MANDATORY
|Award date||2002 May 2|
|Publication status||Published - 2002|
Defence details Date: 2002-05-02 Time: 10:15 Place: Lecture hall D, Solvegatan 39, Lund External reviewer(s) Name: Roberts, Michael R Title: [unknown] Affiliation: Dep. of Biological Sciences, IENS, Lancaster University --- 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 Article: Svennelid F, Sehnke P, Alsterfjord M, Rosenquist M, Larsson H, Ferl RJ, Larsson C, and Sommarin MFusicoccin induces expression of specific plasma membrane H+‑ATPase and 14‑3‑3 isoforms in Arabidopsis. Manuscript