Discussion
In IMAC assay, 12 proteins were identified as Fe2+/Cu2+/Zn2+ binding proteins, including mitochondrial voltage dependent anion channels (VDAC). UpVDAC showed binding abilities to all the three metal ions. His204Ala (H204A) and H219A mutated UpVDAC proteins lost their metal binding ability, and became insensitive to metal-catalyzed oxidation (MCO) induced carbonylation. The overexpression of wild-type UpVDAC made yeast cells more sensitive to oxidative stress, retarded the growth of Arabidopsis seedlings and accelerated the seed aging, while overexpression of mutated UpVDAC weakened these effects of VDAC. These results reveal the relationship between the metal binding ability and carbonylation modification, as well as the probable function of VDAC in regulating cell vitality, seedling growth and seed aging.
Methods
Our previous proteome study found that 13 mitochondria proteins underwent carbonylation modification during the aging of Ulmus pumila L. (Up) seeds. This study detected metal binding proteins through immobilized metal affinity chromatography (IMAC), indicating that metal binding proteins in mitochondria are the main targets of carbonization during seed aging. Biochemistry, molecular and cellular biology methods were adopted to detect metal-protein binding, protein modification and subcellular localization. Yeast and Arabidopsis were used to investigate the biological functions in vivo.