Cloning and functional expression of a gene encoding a vacuolar-type proton-translocating pyrophosphatase from Trypanosoma cruzi.
Acidocalcisomes are acidic Ca(2+)-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.
Hill
J E
JE
Laboratory of Molecular Parasitology, Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802, USA.
Scott
D A
DA
Luo
S
S
Docampo
R
R
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D015698:Genomic Library; D043564:Inorganic Pyrophosphatase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D010944:Plants / Q000201:enzymology; D011522:Protons; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014617:Vacuoles / Q000201:enzymology*
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011522:Protons; D012333:RNA, Messenger; D004024:Dicyclohexylcarbodiimide; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D005033:Ethylmaleimide