Cloning and direct G-protein regulation of phospholipase D from tobacco

Phospholipase D (PLD) and heterotrimeric G-proteins are involved in plant signal transduction pathways at the plasma membrane. There is evidence suggesting that PLD acts downstream from CT-proteins, but a direct interaction of specific members has not been shown. In the present paper, a PLD cDNA clone was isolated from tobacco, expressed as a GST fusion in bacteria, and the recombinant protein was purified by glutathione affinity. Its enzymatic properties identified it as an alpha -type PLD. The alpha -subunit of a G-protein from tobacco was isolated in a similar way. Both proteins were functional in biochemical assays. When the G-protein was included in the PLD assay, a strong dosage-dependent inhibition of the PLD activity was observed. Different control proteins did not exhibit this inhibitory effect. When GST-NtGP alpha1 was activated by incubation with GTP gammaS the inhibitory activity was greatly reduced. These results provide a first indication for a direct regulation of PLD alpha by a heterotrimeric G-protein alpha -subunit in plants. (C) 2001 Elsevier Science B.V. All rights reserved.