CHARACTERIZATION OF A PLASMA MEMBRANE-ASSOCIATED PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE-C FROM SOYBEAN

Phosphoinositide specific phospholipase C (PI-PLC) is a key signal tra nsducing enzyme which generates the second messengers inositol trispho sphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) e ncoding a phosphoinositide specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma m embrane) serum. Genomic DNA gel blot analysis suggested that the corre sponding gene is a member of a multigene family. The deduced amino aci d sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian delta-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian delta-type PI-PLCs and yeast PI-PLC1, the putative Ca2+-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y d omains, and the hi-terminal region is truncated. FLAG epitope-tagged P I-PLC1 fusion protein purified from transgenic tobacco plants showed p hosphoinositide-specific phospholipase C activity. Heterologous expres sion of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant co mplemented the lethality phenotype of haploid PI-PLC1 disruptants. Imm unoblot analysis of the cell fractions prepared from transgenic tobacc o plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protei n indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.