H+-ATPase

The proton pump-ATPase (H+-ATPase) is a major enzyme of the plant plasma membrane, where it couples ATP hydrolysis to the transport of protons out of the cell, thus creating a pH and potential gradient across the membrane. This, in turn, powers secondary transporters which move ions and metabolites across the membrane. In plants, this activity is involved in various markedly different functions or contexts, such as mineral nutrition driven by root tissues, sugar and amino acid transport in the phloem, control of stomata aperture and gas exchange with the external medium, acidification of the external medium for cell expansion,...

H+-ATPase gene family
The plasma membrane H+-ATPases are encoded by a family of at least nine PMA (plasma membrane H+-ATPase) genes in Nicotiana plumbaginifolia. Their transcriptional expression has been monitored using the ß-glucuronidase reporter gene introduced into transgenic plants. PMA2 and PMA4, belonging to two different subfamilies, are the most widely expressed genes. For their detailed enzymatic characterization, the two major H+-ATPase isoforms, PMA2 and PMA4, were expressed in the yeast, Saccharomyces cerevisiae, in which transport functions are also supported by an H+-ATPase. Both N. plumbaginifolia PMA2 and PMA4 were able to functionally replace the yeast H+-ATPase. However, they displayed distinct enzymatic properties.

Protein regulation
A detailed understanding of the molecular mechanisms regulating these various H+-ATPase isoforms has been undertaken. We found that H+-ATPase can be activated by phosphorylation of the penultimate residue, a Thr. This results in displacement of the auto-inhibitory C-terminal domain and subsequent binding of regulatory 14-3-3 proteins. This result in the formation of an hexamer (six H+-ATPases and six 14-3-3 proteins) whose structure has been determined by electron microscopy.
Both PMA2 and PMA4 were provided with a 6-His tag and expressed in transgenic culture cells and plants so that the phosphorylation status, 14-3-3 binding, and enzyme activity can be determined for each isoform. Standard growth conditions will be compared to those in which a particular stress, such as salt and pH stress, or a hormonal treatment is imposed.
Meanwhile two other H+-ATPase phosphorylation sites were discovered and are being characterized.

Physiological roles
In order to study more precisely the respective roles of PMA2 and PMA4 in the plant, we produced transgenic plants that over- or down-express each isoform. Plants in which PMA4 was not expressed were severely affected in that their stomata remained closed, photosynthesis was reduced, sugar loading in the phloem vessels and transport to the non-photosynthetic parts were slowed down and plant development was retarded. We also expressed a constitutively activated H+-ATPase (PMA4) deleted of its auto-inhibitory domain. Transgenic plants had a higher proton pumping activity and lower apoplastic pH. Leaves were epinastic and the stem twisted, suggesting disturbance of cell elongation and thus supporting the acid-growth theory. In addition, the plants were more resistant to salt stress, indicating the involvement of H+-ATPase in activating a plasma membrane Na+/H+antiport.

Key publications

Dambly, S. and Boutry, M. (2001) The two major plasma membrane H+-ATPases display different regulatory properties. J. Biol. Chem. 276, 7017-7022

Arango, M., Gévaudant, F., Oufattole, M., Boutry, M. (2003) The plasma membrane proton pump ATPase: the significance of gene subfamilies, Planta 216, 355-365

Lefebvre, B., Boutry, M., Morsomme, P. (2003) The yeast and plant plasma membrane H+-pump ATPase: divergent regulation for the same function, Prog. Nucleic Acid Res. 74, 203-237

Lefebvre, B., Batoko, H., Duby, G., Boutry, M. (2004) Targeting of a Nicotiana plumbaginifolia H+-ATPase to the plasma membrane is not by default pathway and requires cytosolic structural determinants, Plant Cell 16, 1772-1789

Lefebvre, B., Arango, M., Oufattole, M., Crouzet, J., Purnelle, B. and Boutry, M. (2005) Identification of a Nicotiana plumbaginifolia plasma membrane H+-ATPase gene expressed in the pollen tube. Plant Mol. Biol. 58, 775-787

Kanczewska, J., Marco, S., Vandermeeren, C., Maudoux, O., Rigaud, J., Boutry, M. (2005) Activation of the plant plasma membrane H+-ATPases by phosphorylation and binding of 14-3-3 proteins converts a dimer into an hexamer, Proc. Natl. Acad. Sci. USA, 102, 11675-11680