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Characterization of a plasma membrane‐associated phosphoinositide‐specific phospholipase C from soybean
Author(s) -
Shi Jinrui,
Gonzales Robert A.,
Bhattacharyya Madan K.
Publication year - 1995
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1995.08030381.x
Subject(s) - complementary dna , phospholipase c , signal peptide , biology , inositol , biochemistry , microbiology and biotechnology , subcellular localization , peptide sequence , cytoplasm , gene , signal transduction , receptor
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 δ‐type PI‐PLCs, Dictyostelium discoideum PI‐PLC and yeast PI‐PLC1 in terms of the arrangement of the conserved region. Unlike mammalian δ‐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.