A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast
Résumé
Vacuolar H+-ATPases (V-ATPases) are a specific class of multisubunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localises with the V-ATPase occurs in plants and many protists: the single-subunit H+-translocating inorganic pyrophosphatase (H+-PPase). Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. Here, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1 that is preferentially targeted to internal membranes of yeast alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1 null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalised the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate by itself physiologically suitable internal pH gradients. Besides, they envisage new ways of engineering macrolide drugs tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.
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