Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum, are differentially regulated by phosphorylation of their penultimate threonine.
(2010) The Plant Journal — Vol. 62, n° 2, p. 291-301 (2010)
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- Summary PMA2 and PMA4 are the most widely expressed plasma membrane H(+)-ATPases 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 N. tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, allowing their individual purification. Using these tools, we showed that phosphorylation of the penultimate Thr of both PMA 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 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 one-third of PMA2 or 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, further support for these two H(+)-ATPase subfamilies having different properties.
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Bobik, K., Duby, G., Nizet, Y., Vandermeeren, C., Stiernet, P., Kanczewska, J., & Boutry, M. (2010). Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum, are differentially regulated by phosphorylation of their penultimate threonine. The Plant Journal, 62(2), 291-301. https://doi.org/10.1111/j.1365-313X.2010.04147.x (Original work published 2010)