EGY3: homologue of S2P protease located in chloroplasts

The EGY3 protein is a homologue of site-2 proteases, which are intramembrane zinc metalloproteases. EGY3 itself lacks proteolytic activity due to the absence of a zinc-binding motif. Plentiful evidence indicates that such intramembrane 'pseudoproteases' play significant roles in many diverse processes occurring within the cell. However, the physiological functions of EGY3, as well as its subcellular localization, remain unknown. The subcellular localization of EGY3 protein was investigated using Arabidopsis thaliana protoplasts transformed with EGY3-GFP fusion protein, and immunoblot experiments using the total leaf protein extract, as well as highly purified chloroplasts and fractions of stroma, envelope and thylakoid membrane proteins. The physiological role of EGY3 was studied using two A. thaliana mutant lines devoid of EGY3 protein. Chlorophyll a fluorescence measurement was performed and the egy3 mutant sensitivity to photoinhibition was investigated. Additionally, the abundance of thylakoid membrane complexes was established using blue native gel electrophoresis. We present experimental evidence for thylakoid membrane localization of the EGY3 protein. We show that egy3 mutants display increased value of the non-photochemical quenching parameter and significantly slower recovery rate after photoinhibitory treatment. This was associated with a decrease in the level of proteases involved in photosystem II recovery, Deg1 and FtsH2/8.