tandem-high-pressure-freezing-quick-freeze-substitution-plant-tissues

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

University of Tennessee, Knoxville

The proton pump ATPase (H(+)-ATPase) of the plant plasma membrane is regulated by an autoinhibitory C-terminal domain, which can be displaced by phosphorylation of the penultimate Thr residue and the subsequent binding of 14-3-3 proteins. We performed a mass spectrometric analysis of PMA2 (plasma membrane H(+)-ATPase isoform 2) isolated from Nicotiana tabacum suspension cells and identified two new phosphorylated residues in the enzyme 14-3-3 protein binding site: Thr(931) and Ser(938). When PMA2 was expressed in Saccharomyces cerevisiae, mutagenesis of each of these two residues into Asp prevented growth of a yeast strain devoid of its own H(+)-ATPases. When the Asp mutations were individually introduced in a constitutively activated mutant of PMA2 (E14D), they still allowed yeast growth but at a reduced rate. Purification of His-tagged PMA2 showed that the T931D or S938D mutation prevented 14-3-3 protein binding, although the penultimate Thr(955) was still phosphorylated, indicating that Thr(955) phosphorylation is not sufficient for full enzyme activation. Expression of PMA2 in an N. tabacum cell line also showed an absence of 14-3-3 protein binding resulting from the T931D or S938D mutation. Together, the data show that activation of H(+)-ATPase by the binding of 14-3-3 proteins is negatively controlled by phosphorylation of two residues in the H(+)-ATPase 14-3-3 protein binding site. The data also show that phosphorylation of the penultimate Thr and 14-3-3 binding each contribute in part to H(+)-ATPase activation.

Activation of plant plasma membrane H+-ATPase by 14-3-3 proteins is negatively controlled by two phosphorylation sites within the H+-ATPase C-terminal region.

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.

Tight regulation of the plasma membrane proton pump ATPase (H (+) -ATPase) is necessary for controlling the membrane potential that energizes secondary transporters. This regulation relies on the phosphorylation of the H (+) -ATPase penultimate residue, a theonine, and the subsequent binding of regulatory 14-3-3 proteins, which results in enzyme activation. Using phospho-specific antibodies directed against the phosphorylable Thr of either PMA2 (Plasma membrane H (+) -ATPase from N. plumbaginifolia) or PMA4, we showed that the kinetics and extent of phosphorylation differ between both isoforms according to the growth or environmental conditions like cold stress. (1) Here, we used phospho-specific antibodies to follow PMA2 Thr phosphorylation upon acidification of the cytosol by incubating N. tabacum BY2 cells with four different weak organic acids. Increased PMA2 phosphorylation was observed for three of them, thus highlighting the role of the H (+) -ATPase in cell pH homeostasis.

Activation of the plasma membrane H (+) -ATPase by acid stress: antibodies as a tool to follow the phosphorylation status of the penultimate activating Thr.

Article	Year
Activation of plant plasma membrane H+-ATPase by 14-3-3 proteins is negatively controlled by two phosphorylation sites within the H+-ATPase C-terminal region. The Journal of biological chemistry\| PubMed ID: 19088078	2009
Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine. The Plant journal : for cell and molecular biology\| PubMed ID: 20128881	2010
Activation of the plasma membrane H (+) -ATPase by acid stress: antibodies as a tool to follow the phosphorylation status of the penultimate activating Thr. Plant signaling & behavior\| PubMed ID: 20404493	2010

Krzysztof Bobik

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