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

Acidocalcisomes are acidic Ca2+-storage organelles found in trypanosomatids that are similar to organelles known historically as volutin granules. Aci dification of these organelles is driven in part by a vacuolar H+-pyrophosp hatase (V-H+-PPase), an enzyme that is also present in plant vacuoles and i n some bacteria. Here, we report the cloning and sequencing of a gene encod ing the acidocalcisomal V-H+-PPase of Trypanosoma cruzi. The protein (T. cr uzi 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 s ensitivity to N-ethylmaleimide and N,N'-dicyclohexylcarbodi-imide. Heterolo gous expression of the cDNA encoding TcPPase in the yeast Saccharomyces cer evisiae produced a functional enzyme. Phylogenetic analysis of the availabl e V-H+-PPase sequences indicates that TcPPase is. nearer to the vascular pl ant cluster and the branch containing Chara, a precursor to land plants, th an 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 tar get for the development of new drugs.