Functional analysis of mammalian phospholipase D enzymes.

Phosphatidylcholine-specific phospholipase D (PLD) hydrolyzes the phosphodiester bond of the phosphatidylcholine to generate phosphatidic acid (PA) and regulates several sub-cellular functions. Mammalian genomes contain two genes encoding distinct isoforms of PLD in contrast to invertebrate genomes that include a single PLD gene. However, the significance of two genes within a genome encoding the same biochemical activity remains unclear. Recently, loss of function in the only PLD gene in Drosophila was reported to result in reduced PA levels and a PA dependent collapse of the photoreceptor plasma membrane due to defects in vesicular transport. Phylogenetic analysis reveals that human PLD1 (hPLD1)is evolutionarily closer to dPLD than humanPLD2 (hPLD2). In this study we expressed hPLD1 and hPLD2 in Drosophila and found that while reconstitution of hPLD1 is able to completely rescue retinal degeneration in a loss of function dPLD mutant,hPLD2 was less effective in its ability to mediate a rescue. Using a newly developed analytical method, we determined the acyl chain composition of PA species produced by each enzyme. While dPLD was able to restore the levels of most PA species in dPLD3.1 cells, hPLD1 and hPLD2 each were unable to restore the levels of a subset of unique species of PA. Finally, we found that in contrast to hPLD2, dPLD and hPLD1 are uniquely distributed to the sub-plasma membrane region in photoreceptors. In summary hPLD1 likely represents the ancestral PLD in mammalian genomes while hPLD2 represents neofunctionalization to generate PA at distinct sub-cellular membranes.