Abstract
Mg-chelatase composed by CHLI, CHLD, and CHLH subunits catalyzes the insertion of Mg2+ into protoporphyrin IX. Mg-chelatase is regulated by binding of GUN4 to the catalytic subunit CHLH, the mutants of which resulted in gun phenotype . Here, we reconstituted the Mg-chelatase activity using pea chloroplasts subfractions and Arabidopsis recombinant proteins. Intact pea chloroplasts were separated into soluble (S) and membrane (LM) fractions. Combination of them successfully reconstituted the activity with Mg2+- and DTT-dependent manner. Since plastidic thioredoxin failed to enhance the activity, reduction of other subunits than CHLI may be necessary for the activation. In the absence of Mg2+, all subunits were detected in S, while CHLH and GUN4 transferred to LM in the presence of >5 mM Mg2+. Under this condition, addition of recombinant CHLH and GUN4 to S reconstituted the activity. With this system, we are analyzing the regulation of Mg-chelatase and its correlation with gun phenotype.
