Abstract
Mg-chelatase is composed of three subunits, CHLI, CHLD and CHLH. Mg-insertion by Mg-chelatase requires ATP hydrolysis attributed by CHLI, which primary determines the rate of Mg-insertion. Recently, proteomic analysis of thioredoxin (Trx)-target proteins identified CHLI as one of the candidates (Balmer et al. PNAS, 100, 370, 2003). In fact, cystein residues are conserved in CHLI among higher plants, suggesting that the ATPase activity of CHLI is controlled by Trx. To investigate the Trx-dependent regulation of Mg-chelatase activity, we examined the effect of Trx on ATPase activity of CHLI1 subunit of Arabidopsis thaliana.
Recombinant CHLI1 with His-tag was oxidized, incubated with DTT, reacted with AMS, and separated on SDS-PAGE. This way we could detect DTT-dependent reduction of CHLI1. Trx assisted this reduction. Trx-dependent increase of ATPase activity of CHLI1 was also observed, suggesting that CHLI is one of Trx-target proteins, and its ATPase activity is regulated by Trx.
