Lokalisierung und physiologische Charakterisierung der Serinprotease DEG7 in Arabidopsis thaliana

Abstract

DEG/HtrA proteases are ATP-independent serine endopeptidases and occur in all domains of life. Members of this family contain a chymotrypsin type protease domain with His-Asp-Ser as a catalytic triad and one or more C-terminally located protein-protein interaction PDZ domains. DEG/HtrA proteases are often involved in protein quality control which is especially important in photosynthetic, sessile organisms that are subjected to numerous stress situations, which lead to damaged proteins. The genome of Arabidopsis thaliana encodes for sixteen DEG proteases which are localized in various subcellular compartments.The serine protease DEG7 is twice as long as all other fifteen DEG proteases in A. thaliana, suggesting that DEG7 underwent an internal gene duplication event. The domain arrangement of DEG7 is unusual because it contains two protease domains, one active and one degenerated. Recently, it was reported that DEG7 is located in the chloroplast of A. thaliana and is potentially involved in the PSII repair cycle. I demonstrate here that DEG7 was found in the nucleus, both by localisation studies using DEG7-GFP and detection of DEG7-GFP in isolated nuclei by immunoblotting.In this work, two peptide antibodies against DEG7 were generated, which detected recombinant DEG7 expressed in E. coli. However, both antisera showed poor sensitivity or specificity for DEG7 in plant extracts. Therefore one antiserum was purified by negative and positive adsorption, resulting in an increased specificity for DEG7 in plant extract. However, a second unknown nuclear protein with a similar molecular weight of DEG7 is still detected.The yeast ortholog of DEG7, Nma111p, is localised in the nucleus and mediates apoptosis. The introduction of A. thaliana DEG7 protease into S. cerevisiae demonstrated that overexpression of DEG7 had the same promoting effect on the induction of apoptosis under heat stress as overexpression of Nma111p in the yeast deletion strain ∆nma111. Apoptosis in yeast cells is a process similar to plant programmed cell death (PCD). After the characterisation of two independent ∆deg7 knockout plants, the sensitivity of seedlings of ∆deg7 knockout plants against the fungal toxin Fumonisin B1 as elicitor for programmed cell death was significantly diminished in comparison to seedlings of wildtype plants. I conclude that DEG7 exhibits a pro-apoptotic function and propose that DEG7 acts as a positive regulator of plant PCD by degrading inhibitors of PCD in the nucleus.