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Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases

Perez-Castineira, J.R., Lopez-Marques, R.L., Villalba, J.M., Losada, M., Serrano, A.
Proceedings of the National Academy of Sciences of the United States of America 2002 v.99 no.25 pp. 15914-15919
Saccharomyces cerevisiae, mutants, inorganic pyrophosphatase, Arabidopsis thaliana, genes, bacteria, enzyme activity, ion transport, hydrogen ions, gene expression, messenger RNA, genetic complementation, plasma membrane, Chloroflexus aurantiacus
Two types of proteins that hydrolyze inorganic pyrophosphate (PP(i)), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping pyrophosphatases (sPPases and H(+)-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PP(i) releasing heat, whereas H(+)-PPases, so far unidentified in animal and fungal cells, couple the energy of PP(i) hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H(+)-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H(+)-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PP(i) is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H(+)-PPases can mediate net hydrolysis of PP(i) in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure-function relationships of this type of proton pumps.