PARENT SESSION
Posters P5C Biosynthesis and assembly: Pigments. Abstracts (643-659)

Effects of the gun5 and gun4 mutations on in vitro magnesium chelatase activity in Synechocystis. Paul Davison^*,1, John Gray², Neil Hunter¹, ¹ Dept of Molecular Biology and Biotechnology, Sheffield, UK² Dept of Plant Sciences, Cambridge, UK

ABSTRACT- The gun (genome uncoupled) mutants in Arabidopsis thaliana show a phenotype in which signalling between the nucleus and chloroplast is disrupted, resulting in the expression of nuclear-encoded photosynthetic genes that are normally switched off in wild-type plants in the absence of chloroplasts. Two such mutants, gun4 and gun5, also exhibit pale phenotypes indicating a disruption in chlorophyll biosynthesis. The gun5-1 and cch Arabidopsis mutants each carry single point mutations in the H subunit of the magnesium chelatase enzyme. Magnesium chelatase consists of three subunits (H, I, and D) and catalyses the conversion of protoporphyrin IX to magnesium protoporphyrin IX, the first commited step of chlorophyll biosynthesis; the H subunit is involved in the binding of the porphyrin substrate. To further investigate these mutations they were introduced into the wild-type Synechocystis H subunit and the mutant proteins overexpressed and purified for use in an in vitro magnesium chelatase assay set up for Synechocystis. Tryptophan fluorescence quenching studies on the gun5-1 and cch proteins mixed with deuteroprotoporphyrin IX (a more water soluble substrate than protoporphyrin IX) revealed there was no change in the ability of the mutant proteins to bind substrate. However when these proteins were used in the in vitro chelatase assay with pure Synechocystis I and D subunits, deutero IX and Mg-ATP no discernible activity could be observed. The gun4-1 Arabidopsis mutant carries a single point mutation in a gene of unassigned function. The Synechocystis wild-type GUN4 homologue was isolated and the protein overexpressed and purified and found to bind deutero IX. When GUN4 was added to an in vitro assay containing wild-type H, I and D chelatase activity increased 2-3 fold. In addition, when wild-type H was replaced by gun5-1 or cch protein chelatase activity was restored to almost wild-type levels in the presence of GUN4.

KEY WORDS: gun, magnesium chelatase, Synechocystis