The family of Deg proteases in Arabidopsis thaliana
Zusammenfassung der Projektergebnisse
The protein quality control is important in phototrophic oxygenic organisms that generate large amounts of reactive oxygen species as photosynthetic byproducts that might lead to oxidative damage of proteins. The irreversibly damaged proteins are degraded by a variety of proteolytic enzymes, among them the members of the family of DEG/HtrA proteases. DEG/HtrA serine proteases are ATP-independent enzymes present in all domain of life that combine both, chaperone and protease activities. In this project we performed a biochemical characterization of selected DEG/HtrA proteases from Arabidopsis thaliana and the cyanobacterium Synechocystis sp. PCC6803 with the focus on regulation of proteolytic activity, substrate specificity, formation of oligomeric complexes and their physiological roles in protein quality control and processing. In the past we demonstrated by in vitro studies that the chloroplast DEG2 protease in Arabidopsis performs the proteolytic cleavage of photodamaged D1 protein from photosystem II reaction center. Degradation of photodamaged D1 protein and its rapid replacement by a de novo synthesized functional copy represent an important repair mechanism essential for plant survival under light stress conditions. In this project we investigated a role of DEG2 in the degradation of photodamaged D1 protein in vivo using the reverese genetic approach. The results of this study led us to the conclusion that DEG2 is a part of a larger network of enzymes that ensure protein quality control in photosystem II. We further demonstrated that DEG7 and DEG9 are nuclear proteins. Among the members of the DEG/HtrA family, the DEG7 protease is unique since it contains two protease domains (one active and one degenerated) and four protein-protein interaction PDZ domains. Using yeast-2-hybrid assay and biochemical analysis we demonstrated that DEG7 forms homotrimeric complexes, showing a new principle of oligomerization based on interactions of degenerated protease domains. We proposed that during evolution, a duplicated active protease domain degenerated and acquired a new function in protein-protein interaction. A set of identified potential substrates/interaction partners and mutant analysis suggest that DEG7 might be involved in programmed cell death processes in plants. We further demonstrated that DEG9 is targeted to the nucleolus as the only protease present in this compartment. Mutant analysis revealed that DEG9 might play a role in quality control of proteins involved in ribosomal transcription, processing and modification of ribosomal RNA, or formation of pre-ribosomal particles. We showed that DEG15 is a peroxisomal protease that processes the N-terminal peroxisomal targeting signal PTS2 in nuclear-encoded proteins targeted to peroxisomes. Arabidopsis mutants lacking DEG15 showed an altered phenotype potentially linked to reduced βoxidation, indicating for the first time the impact of protein processing on peroxisomal functions in higher eukaryotes. Finally, we compared three proteases, HtrA, HhoA and HhoB, from Synechocystis and showed that they differ in respect to substrate cleavage sites, temperature and pH optima implying mechanistic differences in comparison with each other. All three proteases formed different homooligomeric complexes with and without substrate and the deletion of the PDZ domain prevented substrate-induced formation of complexes higher than trimers. Our data suggest that each of the three enzymes might contribute differently to the overall proteolytic potential with varying environmental conditions.
Projektbezogene Publikationen (Auswahl)
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(2005). The family of Deg proteases in cyanobacteria and chloroplasts of higher plants. Physiol Plant 123, 413-420
Huesgen PF, Schuhmann H and Adamska I
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(2006). Photodamaged D1 protein is degraded in Arabidopsis mutants lacking the Deg2 protease. FEBS Lett 580, 6929-6932
Huesgen P, Schuhmann H and Adamska I
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(2006). Proteolysis in plant mitochondria and plastids. In: Advances in Plant Physiology, ed. A. Hemantaranjan, Scientific Publishers (India), Jodhpur, vol. 9, pp. 255-294, ISBN 81-7233-459-1
Huesgen PF, Schuhmann H and Adamska I
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(2007). Dual specificites of the glyoxysomal/peroxisomal processing protease Deg15 in higher plants. Proc Natl Acad Sci USA 104, 11501-11506
Helm M, Lück C, Prestele J, Hierl G, Huesgen PF, Fröhlich T, Arnold GJ, Adamska I, Görg A, Lottspeich F and Gietl C
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(2007). The serine protease HhoA from Synechocystis sp. PCC6803: Substrate specificity and formation of a hexameric complex are regulated by the PDZ domain. J Bacteriol 189, 6611-6618
Huesgen PF, Scholz P and Adamska I
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(2008). The DEG15 serine protease cleaves peroxisomal targeting signal 2-containing proteins in Arabidopsis. Plant Physiol 148, 1847-1856
Schuhmann H, Huesgen PF, Gietl C and Adamska I
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(2009). Deg/HtrA proteases as components of a network for photosystem II quality control in chloroplasts and cyanobacteria. Res Microbiol 160, 726-732
Huesgen PF, Schuhmann H and Adamska I
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(2009). The eukaryotic GCP1 is a conserved mitochondrial protein required for progression of embryo development beyond the globular stage in Arabidopsis thaliana. Biochem J 423, 333-341
Haußühl K, Huesgen PF, Meier M, Dessi P, Glaser E, Adamski J and Adamska I
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(2011). A new principle of oligomerization of plant DEG7 protease based on interactions of degenerated protease domains. Biochem J 435, 167-174
Schuhmann H, Mogg U and Adamska I
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(2011). Deg proteases and their role in protein quality control and processing in different subcellular compartments of the plant cell. Physiol Plant, Oct 18 (Epub ahead of print)
Schuhmann H and Adamska I
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(2011). Recombinant Deg/HtrA proteases from Synechocystis sp. PCC6803 differ in substrate specificity, biochemical characteristics and mechanism. Biochem J 435, 733- 742
Huesgen PF, Miranda H, Lam XT, Perthold M, Schuhmann H, Adamska I and Funk C
