title: Proteomic approaches to identify substrates of the three Deg/HtrA proteases of the cyanobacterium Synechocystis sp. PCC 6803.
abstract: The family of Deg/HtrA proteases plays an important role in quality control of cellular proteins in a wide range of organisms. In the genome of the cyanobacterium Synechocystis sp. PCC 6803, a model organism for photosynthetic research and renewable energy products, three Deg proteases are encoded, termed HhoA, HhoB and HtrA. In the present study, we compared wild-type (WT) Synechocystis cells with the single insertion mutants ΔhhoA, ΔhhoB and ΔhtrA. Protein expression of the remaining Deg/HtrA proteases was strongly affected in the single insertion mutants. Detailed proteomic studies using DIGE (difference gel electrophoresis) and N-terminal COFRADIC (N-terminal combined fractional diagonal chromatography) revealed that inactivation of a single Deg protease has similar impact on the proteomes of the three mutants; differences to WT were observed in enzymes involved in the major metabolic pathways. Changes in the amount of phosphate permease system Pst-1 were observed only in the insertion mutant ΔhhoB. N-terminal COFRADIC analyses on cell lysates of ΔhhoB confirmed changed amounts of many cell envelope proteins, including the phosphate permease systems, compared with WT. In vitro COFRADIC studies were performed to identify the specificity profiles of the recombinant proteases rHhoA, rHhoB or rHtrA added to the Synechocystis WT proteome. The combined in vivo and in vitro N-terminal COFRADIC datasets propose RbcS as a natural substrate for HhoA, PsbO for HhoB and HtrA and Pbp8 for HtrA. We therefore suggest that each Synechocystis Deg protease protects the cell through different, but connected mechanisms.
authors: Department of Chemistry, Umeå University, S-90187 Umeå, Sweden.
authors: Lam X
authors: LX
authors: Tam
authors: Department of Chemistry, Umeå University, S-90187 Umeå, Sweden.
authors: Harald
authors: H
authors: Aigner
authors: Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
authors: Evy
authors: E
authors: Timmerman
authors: Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
authors: Kris
authors: K
authors: Gevaert
authors: Department of Chemistry, Umeå University, S-90187 Umeå, Sweden christiane.funk@umu.se.
authors: Christiane
authors: C
authors: Funk
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D057825:Two-Dimensional Difference Gel Electrophoresis
chemical_list: D001426:Bacterial Proteins; C066496:DegS protein, Bacteria; D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; D028061:Phosphate Transport Proteins; D045332:Photosystem II Protein Complex; D021122:Protein Subunits; D011994:Recombinant Proteins; C054266:photosystem II manganese-stabilizing protein; C055831:phosphate permease; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C061682:DegP protease; D012697:Serine Endopeptidases; D012273:Ribulose-Bisphosphate Carboxylase
keywords: N-terminal combined fractional diagonal chromatography (COFRADIC); Synechocystis 6803; difference gel electrophoresis (DIGE); expression; proteases