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

摘要

pAcidocalcisomes are acidic Casup2+/sup-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 Hsup+/sup-pyrophosphatase (V-Hsup+/sup-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-Hsup+/sup-PPase of iTrypanosoma cruzi/i. The protein (iT. cruzi/i 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-Hsup+/sup-PPases were present in TcPPase, explaining its sensitivity to iN/i-ethylmaleimide and iN/i,iN/i′-dicyclohexylcarbodi-imide. Heterologous expression of the cDNA encoding TcPPase in the yeast iSaccharomyces cerevisiae/i produced a functional enzyme. Phylogenetic analysis of the available V-Hsup+/sup-PPase sequences indicates that TcPPase is nearer to the vascular plant cluster and the branch containing iChara/i, a precursor to land plants, than to any of the other pyrophosphatase sequences included in the analysis. The apparent lack of such a V-Hsup+/sup-PPase in mammalian cells may provide a target for the development of new drugs./p