Cloning and functional expression of a gene encoding a vacuolar-type proton-translocating pyrophosphatase from Trypanosoma cruzi

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Abstract
Acidocalcisomes are acidic Ca2+-storage organelles found in trypanosomatids that are similar to organelles known historically as volutin granules. Acidification of these organelles is driven in part by a vacuolar H+-pyrophosphatase (V-H+-PPase), an enzyme that is also present in plant vacuoles and in some bacteria. Here, we report the cloning and sequencing of a gene encoding the acidocalcisomal V-H+-PPase of Trypanosoma cruzi. The protein (T. cruzi pyrophosphatase, TcPPase) predicted from the nucleotide sequence of the gene has 816 amino acids and a molecular mass of 85kDa. Several sequence motifs found in plant V-H+-PPases were present in TcPPase, explaining its sensitivity to N-ethylmaleimide and N,N´-dicyclohexylcarbodi-imide. Heterologous expression of the cDNA encoding TcPPase in the yeast Saccharomyces cerevisiae produced a functional enzyme. Phylogenetic analysis of the available V-H+-PPase sequences indicates that TcPPase is nearer to the vascular plant cluster and the branch containing Chara, a precursor to land plants, than to any of the other pyrophosphatase sequences included in the analysis. The apparent lack of such a V-H+-PPase in mammalian cells may provide a target for the development of new drugs.