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

被引:92
作者
SHI, JR
GONZALES, RA
BHATTACHARYYA, MK
机构
[1] Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, 73402
关键词
D O I
10.1046/j.1365-313X.1995.08030381.x
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
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 Ca2+-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the hi-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.
引用
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页码:381 / 390
页数:10
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