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Two widely expressed plasma membrane H + ‐ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine
Author(s) -
Bobik Krzysztof,
Duby Geoffrey,
Nizet Yannick,
Vandermeeren Caroline,
Stiernet Patrick,
Kanczewska Justyna,
Boutry Marc
Publication year - 2010
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2010.04147.x
Subject(s) - phosphorylation , nicotiana tabacum , gene isoform , biology , protein phosphorylation , atpase , biochemistry , threonine , nicotiana , microbiology and biotechnology , gene , protein kinase a , enzyme , solanaceae , serine
Summary 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.