RESUMO
Allotopic expression is the functional transfer of an organellar gene to the nucleus, followed by synthesis of the gene product in the cytosol and import into the appropriate organellar sub compartment. Here, we focus on mitochondrial genes encoding OXPHOS subunits that were naturally transferred to the nucleus, and critically review experimental evidence that claim their allotopic expression. We emphasize aspects that may have been overlooked before, i.e., when modifying a mitochondrial gene for allotopic expressionâbesides adapting the codon usage and including sequences encoding mitochondrial targeting signalsâthree additional constraints should be considered: (i) the average apparent free energy of membrane insertion (µΔGapp) of the transmembrane stretches (TMS) in proteins earmarked for the inner mitochondrial membrane, (ii) the final, functional topology attained by each membrane-bound OXPHOS subunit; and (iii) the defined mechanism by which the protein translocator TIM23 sorts cytosol-synthesized precursors. The mechanistic constraints imposed by TIM23 dictate the operation of two pathways through which alpha-helices in TMS are sorted, that eventually determine the final topology of membrane proteins. We used the biological hydrophobicity scale to assign an average apparent free energy of membrane insertion (µΔGapp) and a "traffic light" color code to all TMS of OXPHOS membrane proteins, thereby predicting which are more likely to be internalized into mitochondria if allotopically produced. We propose that the design of proteins for allotopic expression must make allowance for µΔGapp maximization of highly hydrophobic TMS in polypeptides whose corresponding genes have not been transferred to the nucleus in some organisms.
Assuntos
Mitocôndrias , Proteínas de Saccharomyces cerevisiae , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Genes Mitocondriais , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mitochondrial F1FO-ATP synthase plays a key role in cellular bioenergetics; this enzyme is present in all eukaryotic linages except in amitochondriate organisms. Despite its ancestral origin, traceable to the alpha proteobacterial endosymbiotic event, the actual structural diversity of these complexes, due to large differences in their polypeptide composition, reflects an important evolutionary divergence between eukaryotic lineages. We discuss the effect of these structural differences on the oligomerization of the complex and the shape of mitochondrial cristae.
Assuntos
Glicogênio Sintase , ATPases Mitocondriais Próton-Translocadoras , Trifosfato de Adenosina/metabolismo , Glicogênio Sintase/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismoRESUMO
The F1 Fo -ATP synthase, a widely distributed nanomotor responsible of ATP synthesis, rotates its central rotor reversibly: In the clockwise direction when viewed from the Fo (with the observer facing the positive side of the energy transducing membrane and looking down into the negative side of the membrane), it functions as ATP synthase, while in counterclockwise sense, it operates as a proton-pumping ATP hydrolase. Regulation exerted by naturally occurring inhibitory proteins of the enzyme appears to function by avoiding ATP hydrolysis while preserving ATP synthesis. The work of Liu et al. describes an unbiased, elegant analytical pipeline that provides important insights into the inhibitory role of the ε-subunit of the bacterial F1 Fo -ATP synthase in vivo. We discuss if a gear-shifting versus a pawl-ratchet mechanism may explain the regulatory role of the ε-subunit.
Assuntos
Trifosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Transporte de Íons , Subunidades Proteicas/metabolismoRESUMO
Triosephosphate isomerase (TIM) is an enzyme of the glycolysis pathway which exists in almost all types of cells. Its structure is the prototype of a motif called TIM-barrel or (α/ß)8 barrel, which is the most common fold of all known enzyme structures. The simplest form in which TIM is catalytically active is a homodimer, in many species of bacteria and eukaryotes, or a homotetramer in some archaea. Here we show that the purified homodimeric TIMs from nine different species of eukaryotes and one of an extremophile bacterium spontaneously form higher order aggregates that can range from 3 to 21 dimers per macromolecular complex. We analysed these aggregates with clear native electrophoresis with normal and inverse polarity, blue native polyacrylamide gel electrophoresis, liquid chromatography, dynamic light scattering, thermal shift assay and transmission electron and fluorescence microscopies, we also performed bioinformatic analysis of the sequences of all enzymes to identify and predict regions that are prone to aggregation. Additionally, the capacity of TIM from Trypanosoma brucei to form fibrillar aggregates was characterized. Our results indicate that all the TIMs we studied are capable of forming oligomers of different sizes. This is significant because aggregation of TIM may be important in some of its non-catalytic moonlighting functions, like being a potent food allergen, or in its role associated with Alzheimer's disease.
Assuntos
Agregados Proteicos , Triose-Fosfato Isomerase/metabolismo , Cromatografia Líquida , Biologia Computacional/métodos , Difusão Dinâmica da Luz , Ativação Enzimática , Expressão Gênica , Cinética , Ligação Proteica , Multimerização Proteica , Sensibilidade e Especificidade , Especificidade da Espécie , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/isolamento & purificaçãoRESUMO
Experimentally relocating mitochondrial genes to the nucleus for functional expression (allotopic expression) is a challenging process. The high hydrophobicity of mitochondria-encoded proteins seems to be one of the main factors preventing this allotopic expression. We focused on subunit II of cytochrome c oxidase (Cox2) to study which modifications may enable or improve its allotopic expression in yeast. Cox2 can be imported from the cytosol into mitochondria in the presence of the W56R substitution, which decreases the protein hydrophobicity and allows partial respiratory rescue of a cox2-null strain. We show that the inclusion of a positive charge is more favorable than substitutions that only decrease the hydrophobicity. We also searched for other determinants enabling allotopic expression in yeast by examining the COX2 gene in organisms where it was transferred to the nucleus during evolution. We found that naturally occurring variations at within-membrane residues in the legume Glycine max Cox2 could enable yeast COX2 allotopic expression. We also evidence that directing high doses of allotopically synthesized Cox2 to mitochondria seems to be counterproductive because the subunit aggregates at the mitochondrial surface. Our findings are relevant to the design of allotopic expression strategies and contribute to the understanding of gene retention in organellar genomes.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Genes Mitocondriais , Proteínas de Membrana/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.
Assuntos
Flagelos/metabolismo , Flagelina/metabolismo , Lipoproteínas/metabolismo , Rhodobacter sphaeroides/fisiologia , Western Blotting , Flagelos/fisiologia , Flagelina/genética , Deleção de Genes , Lipoproteínas/deficiência , Mapeamento de Interação de Proteínas , Rhodobacter sphaeroides/genéticaRESUMO
The mitochondrial ATP synthase of Polytomella exhibits a peripheral stalk and a dimerization domain built by the Asa subunits, unique to chlorophycean algae. The topology of these subunits has been extensively studied. Here we explored the interactions of subunit Asa3 using Far Western blotting and subcomplex reconstitution, and found it associates with Asa1 and Asa8. We also identified the novel interactions Asa1-Asa2 and Asa1-Asa7. In silico analyses of Asa3 revealed that it adopts a HEAT repeat-like structure that points to its location within the enzyme based on the available 3D-map of the algal ATP synthase. We suggest that subunit Asa3 is instrumental in securing the attachment of the peripheral stalk to the membrane sector, thus stabilizing the dimeric mitochondrial ATP synthase.
Assuntos
Proteínas de Algas/química , Membrana Celular/química , Clorofíceas/química , ATPases Mitocondriais Próton-Translocadoras/química , Subunidades Proteicas/química , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Clorofíceas/enzimologia , Clorofíceas/genética , Clorofíceas/ultraestrutura , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Rotary ATPases are a family of enzymes that are thought of as molecular nanomotors and are classified in three types: F, A, and V-type ATPases. Two members (F and A-type) can synthesize and hydrolyze ATP, depending on the energetic needs of the cell, while the V-type enzyme exhibits only a hydrolytic activity. The overall architecture of all these enzymes is conserved and three main sectors are distinguished: a catalytic core, a rotor and a stator or peripheral stalk. The peripheral stalks of the A and V-types are highly conserved in both structure and function, however, the F-type peripheral stalks have divergent structures. Furthermore, the peripheral stalk has other roles beyond its stator function, as evidenced by several biochemical and recent structural studies. This review describes the information regarding the organization of the peripheral stalk components of F, A, and V-ATPases, highlighting the key differences between the studied enzymes, as well as the different processes in which the structure is involved.
RESUMO
Deletion of the yeast mitochondrial gene COX2, encoding subunit 2 (mtCox2) of cytochrome c oxidase (CcO), results in a respiratory-incompetent Δcox2 strain. For a cytosol-synthesized Cox2 to restore respiratory growth, it must carry the W56R mutation (cCox2W56R). Nevertheless, only a fraction of cCox2W56R is matured in mitochondria, allowing â¼60% steady-state accumulation of CcO. This can be attributed either to the point mutation or to an inefficient biogenesis of cCox2W56R. We generated a strain expressing the mutant protein mtCox2W56R inside mitochondria which should follow the canonical biogenesis of mitochondria-encoded Cox2. This strain exhibited growth rates, CcO steady-state levels, and CcO activity similar to those of the wild type; therefore, the efficiency of Cox2 biogenesis is the limiting step for successful allotopic expression. Upon coexpression of cCox2W56R and mtCox2, each protein assembled into CcO independently from its genetic origin, resulting in a mixed population of CcO with most complexes containing the mtCox2 version. Notably, the presence of the mtCox2 enhances cCox2W56R incorporation. We provide proof of principle that an allotopically expressed Cox2 may complement a phenotype due to a mutant mitochondrial COX2 gene. These results are relevant to developing a rational design of genes for allotopic expression intended to treat human mitochondrial diseases.
RESUMO
Subunit II of cytochrome c oxidase (Cox2) is usually encoded in the mitochondrial genome, synthesized in the organelle, inserted co-translationally into the inner mitochondrial membrane, and assembled into the respiratory complex. In chlorophycean algae however, the cox2 gene was split into the cox2a and cox2b genes, and in some algal species like Chlamydomonas reinhardtii and Polytomella sp. both fragmented genes migrated to the nucleus. The corresponding Cox2A and Cox2B subunits are imported into mitochondria forming a heterodimeric Cox2 subunit. When comparing the sequences of chlorophycean Cox2A and Cox2B proteins with orthodox Cox2 subunits, a C-terminal extension in Cox2A and an N-terminal extension in Cox2B were identified. It was proposed that these extensions favor the Cox2A/Cox2B interaction. In vitro studies carried out in this work suggest that the removal of the Cox2B extension only partially affects binding of Cox2B to Cox2A. We conclude that this extension is dispensable, but when present it weakly reinforces the Cox2A/Cox2B interaction.
Assuntos
Clorófitas/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/química , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
The algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex V), with a molecular mass of 1600 kDa. Polytomella, lacking both chloroplasts and a cell wall, has greatly facilitated the purification of the algal ATP-synthase. Each monomer of the enzyme has 17 polypeptides, eight of which are the conserved, main functional components, and nine polypeptides (Asa1 to Asa9) unique to chlorophycean algae. These atypical subunits form the two robust peripheral stalks observed in the highly-stable dimer of the algal ATP synthase in several electron-microscopy studies. The topological disposition of the components of the enzyme has been addressed with cross-linking experiments in the isolated complex; generation of subcomplexes by limited dissociation of complex V; detection of subunit-subunit interactions using recombinant subunits; in vitro reconstitution of subcomplexes; silencing of the expression of Asa subunits; and modeling of the overall structural features of the complex by EM image reconstruction. Here, we report that the amphipathic polymer Amphipol A8-35 partially dissociates the enzyme, giving rise to two discrete dimeric subcomplexes, whose compositions were characterized. An updated model for the topological disposition of the 17 polypeptides that constitute the algal enzyme is suggested. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Assuntos
Proteínas de Algas/química , Chlamydomonas reinhardtii/química , Mitocôndrias/química , ATPases Mitocondriais Próton-Translocadoras/química , Subunidades Proteicas/química , Volvocida/química , Proteínas de Algas/genética , Proteínas de Algas/isolamento & purificação , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Expressão Gênica , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/isolamento & purificação , Modelos Moleculares , Peptídeos/química , Peptídeos/genética , Peptídeos/isolamento & purificação , Polímeros/química , Propilaminas/química , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Volvocida/enzimologia , Volvocida/genéticaRESUMO
Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes.
Assuntos
Clorófitas/enzimologia , ATPases Mitocondriais Próton-Translocadoras/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Subunidades ProteicasRESUMO
The F1FO-ATP synthase of the colorless alga Polytomella sp. exhibits a robust peripheral arm constituted by nine atypical subunits only present in chlorophycean algae. The isolated dimeric enzyme exhibits a latent ATP hydrolytic activity which can be activated by some detergents. To date, the kinetic behavior of the algal ATPase has not been studied. Here we show that while the soluble F1 sector exhibits Michaelis-Menten kinetics, the dimer exhibits a more complex behavior. The kinetic parameters (Vmax and Km) were obtained for both the F1 sector and the dimeric enzyme as isolated or activated by detergent, and this activation was also seen on the enzyme reconstituted in liposomes. Unlike other ATP synthases, the algal dimer hydrolyzes ATP on a wide range of pH and temperature. The enzyme was inhibited by oligomycin, DCCD and Mg-ADP, although oligomycin induced a peculiar inhibition pattern that can be attributed to structural differences in the algal subunit-c. The hydrolytic activity was temperature-dependent and exhibited activation energy of 4 kcal/mol. The enzyme also exhibited a hysteretic behavior with a lag phase strongly dependent on temperature but not on pH, that may be related to a possible regulatory role in vivo.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Volvocida/enzimologia , Difosfato de Adenosina/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Dimerização , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Oligomicinas/farmacologia , Proteólise , ATPases Translocadoras de Prótons/antagonistas & inibidoresRESUMO
The cox3 gene, encoding subunit III of cytochrome c oxidase (Cox3) is in mitochondrial genomes except in chlorophycean algae, where it is localized in the nucleus. Therefore, algae like Chlamydomonas reinhardtii, Polytomella sp. and Volvox carteri, synthesize the Cox3 polypeptide in the cytosol, import it into mitochondria, and integrate it into the cytochrome c oxidase complex. In this work, we followed the in vitro internalization of the Cox3 precursor by isolated, import-competent mitochondria of Polytomella sp. In this colorless alga, the precursor Cox3 protein is synthesized with a long, cleavable, N-terminal mitochondrial targeting sequence (MTS) of 98 residues. In an import time course, a transient Cox3 intermediate was identified, suggesting that the long MTS is processed more than once. The first processing step is sensitive to the metalo-protease inhibitor 1,10-ortophenantroline, suggesting that it is probably carried out by the matrix-located Mitochondrial Processing Protease. Cox3 is readily imported through an energy-dependent import pathway and integrated into the inner mitochondrial membrane, becoming resistant to carbonate extraction. Furthermore, the imported Cox3 protein was assembled into cytochrome c oxidase, as judged by the presence of a labeled band co-migrating with complex IV in Blue Native Electrophoresis. A model for the biogenesis of Cox3 in chlorophycean algae is proposed. This is the first time that the in vitro mitochondrial import of a cytosol-synthesized Cox3 subunit is described.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Multimerização Proteica , Volvocida/enzimologia , Transporte Biológico Ativo , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Transporte ProteicoRESUMO
Mitochondrial F1FO-ATP synthase of chlorophycean algae is a complex partially embedded in the inner mitochondrial membrane that is isolated as a highly stable dimer of 1600kDa. It comprises 17 polypeptides, nine of which (subunits Asa1 to 9) are not present in classical mitochondrial ATP synthases and appear to be exclusive of the chlorophycean lineage. In particular, subunits Asa2, Asa4 and Asa7 seem to constitute a section of the peripheral stalk of the enzyme. Here, we over-expressed and purified subunits Asa2, Asa4 and Asa7 and the corresponding amino-terminal and carboxy-terminal halves of Asa4 and Asa7 in order to explore their interactions in vitro, using immunochemical techniques, blue native electrophoresis and affinity chromatography. Asa4 and Asa7 interact strongly, mainly through their carboxy-terminal halves. Asa2 interacts with both Asa7 and Asa4, and also with subunit α in the F1 sector. The three Asa proteins form an Asa2/Asa4/Asa7 subcomplex. The entire Asa7 and the carboxy-terminal half of Asa4 seem to be instrumental in the interaction with Asa2. Based on these results and on computer-generated structural models of the three subunits, we propose a model for the Asa2/Asa4/Asa7 subcomplex and for its disposition in the peripheral stalk of the algal ATP synthase.
Assuntos
Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/química , Peptídeos/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Simulação por Computador , Dimerização , Eletroforese em Gel de Poliacrilamida , Membranas Mitocondriais/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Complexos Multiproteicos , Subunidades Proteicas/biossíntese , Subunidades Proteicas/isolamento & purificação , Volvocida/enzimologiaRESUMO
Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010) [1] transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2(W56R)), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2(W56R) was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2+Cox2(W56R) strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20-25% lower in the enzyme containing Cox2(W56R) than in the one with Cox2(WT). Tandem mass spectrometry confirmed that W(56) was substituted by R(56) in Cox2(W56R). In addition, Cox2(W56R) exhibited the same N-terminus than Cox2(WT), indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2(W56R) during maturation. Thus, Cox2(W56R) is identical to Cox2(WT) except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2(W56R) complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2(W56R) follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2(W56R) restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels.
Assuntos
Citoplasma/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Oxigênio/metabolismo , Mutação Puntual/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Respiração Celular/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Imunoensaio , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Eletroforese em Gel de Poliacrilamida Nativa , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em TandemRESUMO
In most eukaryotes the subunit 2 of cytochrome c oxidase (COX2) is encoded in intact mitochondrial genes. Some green algae, however, exhibit split cox2 genes (cox2a and cox2b) encoding two polypeptides (COX2A and COX2B) that form a heterodimeric COX2 subunit. Here, we analyzed the distribution of intact and split cox2 gene sequences in 39 phylogenetically diverse green algae in phylum Chlorophyta obtained from databases (28 sequences from 22 taxa) and from new cox2 data generated in this work (23 sequences from 18 taxa). Our results support previous observations based on a smaller number of taxa, indicating that algae in classes Prasinophyceae, Ulvophyceae, and Trebouxiophyceae contain orthodox, intact mitochondrial cox2 genes. In contrast, all of the algae in Chlorophyceae that we examined exhibited split cox2 genes, and could be separated into two groups: one that has a mitochondrion-localized cox2a gene and a nucleus-localized cox2b gene ("Scenedesmus-like"), and another that has both cox2a and cox2b genes in the nucleus ("Chlamydomonas-like"). The location of the split cox2a and cox2b genes was inferred using five different criteria: differences in amino acid sequences, codon usage (mitochondrial vs. nuclear), codon preference (third position frequencies), presence of nucleotide sequences encoding mitochondrial targeting sequences and presence of spliceosomal introns. Distinct green algae could be grouped according to the form of cox2 gene they contain: intact or fragmented, mitochondrion- or nucleus-localized, and intron-containing or intron-less. We present a model describing the events that led to mitochondrial cox2 gene fragmentation and the independent and sequential migration of cox2a and cox2b genes to the nucleus in chlorophycean green algae. We also suggest that the distribution of the different forms of the cox2 gene provides important insights into the phylogenetic relationships among major groups of Chlorophyceae.
Assuntos
Núcleo Celular/genética , Clorófitas/genética , Ciclo-Oxigenase 2/genética , Modelos Genéticos , Filogenia , Sequência de Aminoácidos , Sequência de Bases , Análise por Conglomerados , Códon/genética , Biologia Computacional , Primers do DNA/genética , DNA Mitocondrial/genética , Funções Verossimilhança , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
In the vast majority of eukaryotic organisms, the mitochondrial cox2 gene encodes subunit II of cytochrome c oxidase (COX2). However, in some lineages including legumes and chlorophycean algae, the cox2 gene migrated to the nucleus. Furthermore, in chlorophycean algae, this gene was split in two different units. Thereby the COX2 subunit is encoded by two independent nuclear genes, cox2a and cox2b, and mitochondria have to import the cytosol-synthesized COX2A and COX2B subunits and assemble them into the cytochrome c oxidase complex. In the chlorophycean algae Chlamydomonas reinhardtii and Polytomella sp., the COX2A precursor exhibits a long (130-140 residues), cleavable mitochondrial targeting sequence (MTS). In contrast, COX2B lacks an MTS, suggesting that mitochondria use different mechanisms to import each subunit. Here, we explored the in vitro import processes of both, the Polytomella sp. COX2A precursor and the COX2B protein. We used isolated, import-competent mitochondria from this colorless alga. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A seems to follow an energy-dependent import pathway, through which it finally integrates into the inner mitochondrial membrane. In addition, the MTS of the COX2A precursor is eliminated. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved.
Assuntos
Clorófitas/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Multimerização Proteica , Subunidades Proteicas/metabolismo , Aldeído Desidrogenase/metabolismo , Animais , Núcleo Celular/enzimologia , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico , RatosRESUMO
In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e-AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, ΔΨ-independent pathway.