Abstract
Molecular procedures have been applied to isolate plant calmodulin-binding proteins. A petunia cDNA expression library was screened with 35S-labeled recombinant calmodulin as a probe, and a cDNA coding for a Ca(2+)-dependent calmodulin-binding protein was isolated. The deduced amino acid sequence of the petunia protein (500 amino acid residues, 58 kDa) has 67% overall amino acid sequence similarity to glutamate decarboxylase (GAD) from Escherichia coli (466 amino acid residues, 53 kDa). The recombinant protein expressed in E. coli cells displays GAD activity, i.e. catalyzes the conversion of glutamic acid to gamma-aminobutyric acid and binds calmodulin, whereas E. coli GAD does not bind calmodulin. The calmodulin binding domain in the petunia GAD was mapped by binding truncated forms of GAD immobilized on nitrocellulose membranes to recombinant petunia 35S-calmodulin as well as to biotinylated bovine calmodulin and by binding truncated forms of GAD to calmodulin-Sepharose columns. The calmodulin binding domain in petunia GAD is part of a carboxyl end extension that is not present in E. coli GAD. Polyclonal antibodies raised against the recombinant petunia GAD detect a single protein band from plant extracts of gel mobility identical to that of the recombinant GAD. Moreover, the plant protein binds calmodulin in vitro. This is the first report of the isolation of a GAD gene from plants and of a calmodulin-binding GAD from any organism. Our results raise the possibility that intracellular Ca2+ signals via calmodulin are involved in the regulation of gamma-aminobutyric acid synthesis in plants.
Highlights
From the $Department of Plant Genetics, Weizmann Instituteof Science, 76100 Rehouot,Israel and the §National Instituteof Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki305, Japan
Polyclonal antibodies raised We have recently applied a molecular approach to isolate against the recombinant petunia glutamate decarboxylase (GAD) detect a single cDNAs coding for calmodulin-binding proteins by using high protein band from plant extractsof gel mobility iden- specific activity recombinant 35S-labeled calmodulin asa tical to that of the recombinant GAD
Cells were Studier et al (1990).For mapping the calmodulin binding domain of pelleted by 5-min centrifugation a t 2,500 X g at 4 "C, washed with GAD, DNA fragments derived from the GAD cDNA were fused inice-cold 50 mM Tris-HC1, pH 7.5, precipitated as above, and proteins frame to thecoding sequence of glutathione S-transferasein a pGEX
Summary
From the $Department of Plant Genetics, Weizmann Instituteof Science, 76100 Rehouot,Israel and the §National Instituteof Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki305, Japan. We report the isolation and characterization of a cDNA that codes for a calmodulin-binding glutamate decarboxylase (GAD)’ from petunia.
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