A PLANT GLUTAMATE-DECARBOXYLASE CONTAINING A CALMODULIN-BINDING DOMAIN - CLONING, SEQUENCE, AND FUNCTIONAL-ANALYSIS

Molecular procedures have been applied to isolate plant calmodulin-bin ding proteins. A petunia CDNA expression library was screened with S-3 5-labeled recombinant calmodulin as a probe, and a CDNA coding for a C a2+-dependent calmodulin-binding protein was isolated. The deduced ami no acid sequence of the petunia protein (500 amino acid residues, 58 k Da) has 67% overall amino acid sequence similarity to glutamate decarb oxylase (GAD) from Escherichia coli (466 amino acid residues, 53 kDa). The recombinant protein expressed in E. coli cells displays GAD activ ity, i.e. catalyzes the conversion of glutamic acid to gamma-aminobuty ric acid and binds calmodulin, whereas E. coli GAD does not bind calmo dulin. The calmodulin binding domain in the petunia GAD was mapped by binding truncated forms of GAD immobilized on nitrocellulose membranes to recombinant petunia S-35-calmodulin as well as to biotinylated bov ine calmodulin and by binding truncated forms of GAD to calmodulin-Sep harose columns. The calmodulin binding domain in petunia GAD is part o f a carboxyl end extension that is not present in E. coli GAD. Polyclo nal antibodies raised against the recombinant petunia GAD detect a sin gle 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.