Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean.
Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid 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 Ca(2+)-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.
Shi
J
J
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA.
Gonzales
R A
RA
Bhattacharyya
M K
MK
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D018076:DNA, Complementary; D015723:Gene Library; D017343:Genes, Plant; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D008322:Mammals; D008969:Molecular Sequence Data; D005810:Multigene Family; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D015290:Second Messenger Systems; D015398:Signal Transduction; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology
D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase