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 G-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-NtGPalpha1 was activated by incubation with GTPgammaS the inhibitory activity was greatly reduced. These results provide a first indication for a direct regulation of PLDalpha by a heterotrimeric G-protein alpha-subunit in plants.
Lein
W
W
Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, D-06466, Gatersleben, Germany.
Saalbach
G
G
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000737:chemistry; D006150:Guanine Nucleotides / Q000737:chemistry; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D010940:Plant Proteins / Q000378:metabolism*; D010947:Plants, Toxic; D016415:Sequence Alignment; D015398:Signal Transduction; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D006150:Guanine Nucleotides; D010940:Plant Proteins; D005982:Glutathione Transferase; D010739:Phospholipase D; D020962:Heterotrimeric GTP-Binding Proteins