The two major plant plasma membrane H+-ATPases display different regulatory properties

The major plant plasma membrane H+-ATPases fall into two gene categories, s ubfamilies I and II, However, in many plant tissues, expression of the two subfamilies overlaps, thus precluding individual characterization. Yeast ex pression of PMA2 and PMA4, representatives of the two plasma membrane H+-AT Pase subfamilies in Nicotiana plumbaginifolia, has previously shown that (i ) the isoforms have distinct enzymatic properties and that (ii) PMA2 is reg ulated by phosphorylation of its penultimate residue (Thr) and binds regula tory 14-3-3 proteins, resulting in the displacement of the autoinhibitory C -terminal domain. To obtain insights into regulatory differences between th e two subfamilies, we have constructed various chimeric proteins in which t he 110 residue C-terminal-encoding region of PMA2 was progressively substit uted by the corresponding sequence from PMA4, The PMA2 autoinhibitory domai n was localized to a region between residues 851 and 915 and could not be s ubstituted by the corresponding region of PMA4. In contrast to PMA2, PMA4 w as poorly phosphorylated at its penultimate residue (Thr) and bound 14-3-3 proteins weakly. The only sequence difference around the phosphorylation si te is located two residues upstream of the phosphorylated Thr, It is Ser in PMA2 (as in most members of subfamily I) and His in PMA4 (as in most membe rs of subfamily II). Substitution of His by Ser in PMA4 resulted in an enzy me showing increased phosphorylation status, 14-13-3 binding, and ATPase ac tivity, as well as improved yeast growth. The reverse substitution of Ser b y His in PMA2 resulted in the failure of this enzyme to complement the abse nce of yeast H+-ATPases. These results show that the two plant H+-ATPase su bfamilies differ functionally in their regulatory properties.