V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which 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 normalized 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. Also, they suggest new ways of engineering macrolide drug 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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Research Article|
June 28 2011
A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast
José R. Pérez-Castiñeira;
José R. Pérez-Castiñeira
1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Avda. Americo Vespucio, 49, 41092 Sevilla, Spain
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Agustín Hernández;
Agustín Hernández
1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Avda. Americo Vespucio, 49, 41092 Sevilla, Spain
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Rocío Drake;
Rocío Drake
1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Avda. Americo Vespucio, 49, 41092 Sevilla, Spain
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Aurelio Serrano
Aurelio Serrano
1
1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Avda. Americo Vespucio, 49, 41092 Sevilla, Spain
1To whom correspondence should be addressed (email aurelio@ibvf.csic.es).
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Publisher: Portland Press Ltd
Received:
March 10 2011
Revision Received:
May 03 2011
Accepted:
May 05 2011
Accepted Manuscript online:
May 26 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 437 (2): 269–278.
Article history
Received:
March 10 2011
Revision Received:
May 03 2011
Accepted:
May 05 2011
Accepted Manuscript online:
May 26 2011
Citation
José R. Pérez-Castiñeira, Agustín Hernández, Rocío Drake, Aurelio Serrano; A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast. Biochem J 15 July 2011; 437 (2): 269–278. doi: https://doi.org/10.1042/BJ20110447
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