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

Abstract

SUMMARYThe 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.

A Phosphorylation in the C-terminal Auto-inhibitory Domain of the Plant Plasma Membrane H+-ATPase Activates the Enzyme with No Requirement for Regulatory 14-3-3 Proteins

“…Under standard culture conditions for N. tabacum BY2 cells, both Thr-955 phosphorylation and 14-3-3 protein binding are moderate and decrease during culture growth (30). We therefore characterized cells collected at a young stage (i.e.…”

“…We therefore wondered whether this was also the case for the T889D mutant. To assess the level of phosphorylation of the penultimate Thr-955, we used the monoclonal antibody mabPMA2pT, which is specific for PMA2 phosphorylated at Thr-955 (30). Western blotting analysis of the yeast plasma membrane fraction showed a reduction in Thr-955 phosphorylation and 14-3-3 protein binding with the PMA2-Thr889Asp mutant when compared with the wild type (Fig.…”

“…Identification of a New PMA2 Phosphorylation Site-To identify phosphorylation sites in PMA2, one of the two most widely expressed H ϩ -ATPases in N. tabacum, His 6 -tagged PMA2 was expressed in N. tabacum plants (30) and N. tabacum BY2 cell lines (31), solubilized from the microsomal fraction, purified by Ni 2ϩ -NTA chromatography, and analyzed by SDS-PAGE. The PMA2 band was then excised from the gel and digested with trypsin, and the resulting peptides were analyzed by nano LC-MS/MS as described under "Experimental Procedures."…”

Plant Plasma Membrane H+-ATPase in Adaptation of Plants to Abiotic Stresses

“…Two extensively expressed plasma membrane H + -ATPase isoforms of Nicotiana tabacum (PMA2 and PMA4) are differentially regulated by phosphorylation of their penultimate threonine. Cold stress reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were observed (Bobik et al, 2010a). A phosphorylation event requires action of a protein phosphatases to make regulation reversible.…”

Isolation and expression analysis of four members of the plasma membrane H+-ATPase gene family in Hevea brasiliensis

“…PM H ? -ATPases are activated by phosphorylation of the penultimate residue (a threonine) in the C-terminal autoinhibitory domain, which is an important post-translational modification, and the subsequent binding of regulatory 14-3-3 proteins (Morsomme and Boutry 2000;Bobik et al 2010).…”