RESUMO
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the fixation of CO2 in photosynthesis. Despite its pivotal role, Rubisco is an inefficient enzyme and thus is a key target for directed evolution. Rubisco biogenesis depends on auxiliary factors, including the GroEL/ES-type chaperonin for folding and the chaperone RbcX for assembly. Here we performed directed evolution of cyanobacterial form I Rubisco using a Rubisco-dependent Escherichia coli strain. Overexpression of GroEL/ES enhanced Rubisco solubility and tended to expand the range of permissible mutations. In contrast, the specific assembly chaperone RbcX had a negative effect on evolvability by preventing a subset of mutants from forming holoenzyme. Mutation F140I in the large Rubisco subunit, isolated in the absence of RbcX, increased carboxylation efficiency approximately threefold without reducing CO2 specificity. The F140I mutant resulted in a â¼55% improved photosynthesis rate in Synechocystis PCC6803. The requirement of specific biogenesis factors downstream of chaperonin may have retarded the natural evolution of Rubisco.
Assuntos
Proteínas de Bactérias/metabolismo , Evolução Molecular Direcionada/métodos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Multimerização Proteica , Ribulose-Bifosfato Carboxilase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Fotossíntese , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Synechococcus/enzimologia , Synechococcus/genética , Synechococcus/metabolismo , Synechocystis/enzimologia , Synechocystis/genética , Synechocystis/metabolismoRESUMO
The membrane-integrated metalloprotease FtsH11 of Arabidopsis thaliana is proposed to be dual-targeted to mitochondria and chloroplasts. A bleached phenotype was observed in ftsh11 grown at long days or continuous light, pointing to disturbances in the chloroplast. Within the chloroplast, FtsH11 was found to be located exclusively in the envelope. Two chloroplast-located proteins of unknown function (Tic22-like protein and YGGT-A) showed significantly higher abundance in envelope membranes and intact chloroplasts of ftsh11 and therefore qualify as potential substrates for the FtsH11 protease. No proteomic changes were observed in the mitochondria of 6-week-old ftsh11 compared with wild type, and FtsH11 was not immunodetected in these organelles. The abundance of plastidic proteins, especially of photosynthetic proteins, was altered even during standard growth conditions in total leaves of ftsh11. At continuous light, the amount of photosystem I decreased relative to photosystem II, accompanied by a drastic change of the chloroplast morphology and a drop of non-photochemical quenching. FtsH11 is crucial for chloroplast structure and function during growth in prolonged photoperiod.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Cloroplastos/fisiologia , Metaloproteases/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Metaloproteases/genética , Metaloproteases/metabolismo , Fenótipo , FotoperíodoRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Periplásmicas/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Serina Endopeptidases/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Synechocystis/enzimologia , Proteínas de Bactérias/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Mutagênese Insercional , Proteínas Periplásmicas/genética , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Complexo de Proteína do Fotossistema II/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteólise , Proteômica/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Serina Endopeptidases/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Especificidade por Substrato , Synechocystis/genética , Synechocystis/metabolismo , Eletroforese em Gel Diferencial BidimensionalRESUMO
FtsHs are a well-characterized family of membrane bound proteases containing an AAA (ATPase associated with various cellular activities) and a Zn(2+) metalloprotease domain. FtsH proteases are found in eubacteria, animals and plants and are known to have a crucial role in housekeeping proteolysis of membrane proteins. In Arabidopsis thaliana, 12 FtsH family members are present (FtsH 1-12) and their subcellular localization is restricted to mitochondria and chloroplasts. In addition, five genes coding for proteins homologous to FtsH (FtsHi 1-5) have been detected in the genome, lacking the conserved zinc-binding motif HEXXH, which presumably renders them inactive for proteolysis. These inactive FtsHs as well as nine of the active FtsHs are thought to be localized in the chloroplast. In this article, we shortly summarize the recent findings on plastidic FtsH proteases in text and figures. We will mainly focus on FtsH 1, 2, 5 and 8 localized in the thylakoid membrane and known for their importance in photosynthesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Tilacoides/enzimologia , Arabidopsis/química , Arabidopsis/enzimologia , Proteínas de Cloroplastos/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Membrana/genética , Fotossíntese , Proteólise , Transcrição GênicaRESUMO
Darwinian fitness analyses were performed, comparing single ftsh mutants with wild-type Arabidopsis thaliana plants grown under controlled laboratory conditions and in the field, by measuring plant size, survival rate, and silique and seed production. Additionally, three genotypes of ΔFtsH6 were analysed, under controlled growth conditions, with respect to both their ability to degrade the light-harvesting complex of photosystem II during senescence and light acclimation. In the field, substantial increases in variegation and reductions in growth were observed in the ΔFtsH2, ΔFtsH5 and ΔFtsH10 mutants; FtsH2 seemed particularly important for plant survival. Despite being grown in relatively cold weather, the ΔFtsH11 mutant displayed strong phenotypic deviations from wild type. Both ΔFtsH10 and ΔFtsH3 mutants exhibited less severe phenotypic changes, but were different from wild-type plants when placed in the field as young plants. When older ΔFtsH3 or ΔFtsH10 mutants were placed outdoors, no phenotypic differences from wild type were observed. Three genotypes of ΔFtsH6 displayed no phenotypic deviations from wild-type plants. Under controlled growth conditions, during senescence and light acclimation, no differences in the amount of chlorophyll or Photosystem II light-harvesting complex b3 (Lhcb3) were detected in ΔFtsH6 mutants compared with the wild type. Therefore, FtsH6 seems to be unimportant for LHCII degradation in vivo.