The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved 
in cardiolipin metabolism, but it is not known why it forms multiple transcripts 
as a result of alternative splicing. Here we studied the intracellular 
localization, enzymatic activity, and metabolic function of four isoforms of 
human tafazzin and three isoforms of Drosophila tafazzin upon expression in 
different mammalian and insect systems. When expressed in HeLa cells, all 
isoforms were localized in mitochondria except for the B-form of Drosophila 
tafazzin, which was associated with multiple intracellular membranes. Among the 
human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 
(Delta5) had transacylase activity, and only these two isoforms were able to 
restore a normal cardiolipin pattern, normal respiratory activity of 
mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Delta5 
were associated with large protein complexes in 293T cell mitochondria, but 
treatment with alkali and proteinase K suggested that the Delta5 isoform was 
more integrated into the hydrophobic core of the membrane than the FL isoform. 
Although all Drosophila isoforms showed transacylase activity in vitro, only the 
A-form supported cardiolipin remodeling in flies. The data suggest that humans 
express two mitochondrial isoenzymes of tafazzin that have similar transacylase 
activities but different membrane topologies. Furthermore, the data show that 
the expression of human tafazzin in flies creates cardiolipin with a Drosophila 
pattern, suggesting that the characteristic fatty acid profile of cardiolipin is 
not determined by the substrate specificity of tafazzin.