Patrice Hamel

Biochemical requirements for the maturation of mitochondrial c-type cytochromes

Cytochromes c are metalloproteins that function in electron transfer reactions and contain a heme moiety covalently attached via thioether linkages between the co-factor and a CXXCH motif in the protein. Covalent attachment of the heme group occurs on the positive side of all energy-transducing membranes (bacterial periplasm, mitochondrial intermembrane space and thylakoid lumen) and requires minimally: 1) synthesis and translocation of the apocytochromes c and heme across at least one biological membrane, 2) reduction of apocytochromes c and heme and maintenance under a reduced form prior to 3) catalysis of the heme attachment reaction. Surprisingly, the conversion of apoforms of cytochromes c to their respective holoforms occurs through at least three different pathways (systems I, II and III). In this review, we detail the assembly process of soluble cytochrome c and membrane-bound cytochrome c1, the only two mitochondrial c-type cytochromes that function in respiration. Mitochondrial c-type cytochromes are matured in the intermembrane space via the system I or system III pathway, an intriguing finding considering that the biochemical requirements for cytochrome c maturation are believed to be common regardless of the energy-transducing membrane under study.

Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases.

Heme attachment to the apoforms of fungal mitochondrial cytochrome c and c1 requires the activity of cytochrome c and c1 heme lyases (CCHL and CC1HL), which are enzymes with distinct substrate specificity. However, the presence of a single heme lyase in higher eukaryotes is suggestive of broader substrate specificity. Here, we demonstrate that yeast CCHL is active toward the non-cognate substrate apocytochrome c1, i.e. CCHL promotes low levels of apocytochrome c1 conversion to its holoform in the absence of CC1HL. Moreover, that the single human heme lyase also displays a broader cytochrome specificity is evident from its ability to substitute for both yeast CCHL and CC1HL. Multicopy and genetic suppressors of the absence of CC1HL were isolated and their analysis revealed that the activity of CCHL toward cytochrome c1 can be enhanced by: 1) reducing the abundance of the cognate substrate apocytochrome c, 2) increasing the accumulation of CCHL, 3) modifying the substrate-enzyme interaction through point mutations in CCHL or cytochrome c1, or 4) overexpressing Cyc2p, a protein known previously only as a mitochondrial biogenesis factor. Based on the functional interaction of Cyc2p with CCHL and the presence of a putative FAD-binding site in the protein, we hypothesize that Cyc2p controls the redox chemistry of the heme lyase reaction.

Lumen Thiol Oxidoreductase1, a Disulfide Bond-Forming Catalyst, Is Required for the Assembly of Photosystem II in Arabidopsis

This study demonstrates that Arabidopsis thaliana Lumen Thiol Oxidoreductase1 (LTO1) catalyzes disulfide bond formation in the thylakoid lumen of the chloroplast. Loss of LTO1 function yields an assembly defect in photosystem II, a photosynthetic complex that requires a disulfide bond in a structural subunit localized in the lumen. Here, we identify Arabidopsis thaliana Lumen Thiol Oxidoreductase1 (LTO1) as a disulfide bond–forming enzyme in the thylakoid lumen. Using topological reporters in bacteria, we deduced a lumenal location for the redox active domains of the protein. LTO1 can partially substitute for the proteins catalyzing disulfide bond formation in the bacterial periplasm, which is topologically equivalent to the plastid lumen. An insertional mutation within the LTO1 promoter is associated with a severe photoautotrophic growth defect. Measurements of the photosynthetic activity indicate that the lto1 mutant displays a limitation in the electron flow from photosystem II (PSII). In accordance with these measurements, we noted a severe depletion of the structural subunits of PSII but no change in the accumulation of the cytochrome b6f complex or photosystem I. In a yeast two-hybrid assay, the thioredoxin-like domain of LTO1 interacts with PsbO, a lumenal PSII subunit known to be disulfide bonded, and a recombinant form of the molecule can introduce a disulfide bond in PsbO in vitro. The documentation of a sulfhydryl-oxidizing activity in the thylakoid lumen further underscores the importance of catalyzed thiol-disulfide chemistry for the biogenesis of the thylakoid compartment.

Cell-specific differences in the requirements for translation quality control

Protein synthesis has an overall error rate of approximately 10-4 for each mRNA codon translated. The fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and accurate selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. To ensure faithful aa-tRNA synthesis, many aaRSs employ a proofreading (“editing”) activity, such as phenylalanyl-tRNA synthetases (PheRS) that hydrolyze mischarged Tyr-tRNAPhe. Eukaryotes maintain two distinct PheRS enzymes, a cytoplasmic (ctPheRS) and an organellar form. CtPheRS is similar to bacterial enzymes in that it consists of a heterotetramer in which the α-subunits contain the active site and the β-subunits harbor the editing site. In contrast, mitochondrial PheRS (mtPheRS) is an α-subunit monomer that does not edit Tyr-tRNAPhe, and a comparable transacting activity does not exist in organelles. Although mtPheRS does not edit, it is extremely specific as only one Tyr-tRNAPhe is synthesized for every ∼7,300 Phe-tRNAPhe, compatible with an error rate in translation of ∼10-4. When the error rate of mtPheRS was increased 17-fold, the corresponding strain could not grow on respiratory media and the mitochondrial genome was rapidly lost. In contrast, error-prone mtPheRS, editing-deficient ctPheRS, and their wild-type counterparts all supported cytoplasmic protein synthesis and cell growth. These striking differences reveal unexpectedly divergent requirements for quality control in different cell compartments and suggest that the limits of translational accuracy may be largely determined by cellular physiology.

Eukaryotic complex I: functional diversity and experimental systems to unravel the assembly process

AbstractWith more than 40 subunits, one FMN co-factor and eight FeS clusters, complex I or NADH:ubiquinone oxidoreductase is the largest multimeric respiratory enzyme in the mitochondria. In this review, we focus on the diversity of eukaryotic complex I. We describe the additional activities that have been reported to be associated with mitochondrial complex I and discuss their physiological significance. The recent identification of complex I-like enzymes in the hydrogenosome, a mitochondria-derived organelle is also discussed here. Complex I assembly in the mitochondrial inner membrane is an intricate process that requires the cooperation of the nuclear and mitochondrial genomes. The most prevalent forms of mitochondrial dysfunction in humans are deficiencies in complex I and remarkably, the molecular basis for 60% of complex I-linked defects is currently unknown. This suggests that mutations in yet-to-be-discovered assembly genes should exist. We review the different experimental systems for the study of complex I assembly. To our knowledge, in none of them, large screenings of complex I mutants have been performed. We propose that the unicellular green alga Chlamydomonas reinhardtii is a promising system for such a study. Complex I mutants can be easily scored on a phenotypical basis and a large number of transformants generated by insertional mutagenesis can be screened, which opens the possibility to find new genes involved in the assembly of the enzyme. Moreover, mitochondrial transformation, a recent technological advance, is now available, allowing the manipulation of all five ncomplex I mitochondrial genes in this organism.

A heme-sensing mechanism in the translational regulation of mitochondrial cytochrome c oxidase biogenesis.

Heme plays fundamental roles as cofactor and signaling molecule in multiple pathways devoted to oxygen sensing and utilization in aerobic organisms. For cellular respiration, heme serves as a prosthetic group in electron transfer proteins and redox enzymes. Here we report that in the yeast Saccharomyces cerevisiae, a heme-sensing mechanism translationally controls the biogenesis of cytochrome c oxidase (COX), the terminal mitochondrial respiratory chain enzyme. We show that Mss51, a COX1 mRNA-specific translational activator and Cox1 chaperone, which coordinates Cox1 synthesis in mitoribosomes with its assembly in COX, isxa0a heme-binding protein. Mss51 contains two heme regulatory motifs or Cys-Pro-X domains located in its N terminus. Using a combination of inxa0vitro and inxa0vivo approaches, we have demonstrated that these motifs are important for heme binding and efficient performance of Mss51 functions. We conclude that heme sensing by Mss51 regulates COX biogenesis and aerobic energy production.

Isolation of an Arabidopsis thaliana cDNA by complementation of a yeast abc1 deletion mutant deficient in complex III respiratory activity

The yeast Abc1 protein acts as a chaperone-like protein essential for the proper conformation and efficient functioning of the respiratory complex III. By functional complementation of a yeast abc1 mutant, we have identified an Arabidopsis thaliana cDNA that corresponds to a single copy gene and encodes a protein sharing 45% similarity with the yeast Abc1p protein. Cytochrome spectra and respiratory activity measurements have shown that the plant protein allows a partial restoration of the complex III activity. No major difference in the steady-state level of ABC1At mRNA was observed in various plant tissues, suggesting that ABC1At is constitutively expressed in A. thaliana. Phylogenetic analysis revealed that the Abc1At protein belongs to a large family of proteins composed of two eukaryotic and one prokaryotic subgroups differing by their degree of similarity and probably by their function.

Apigenin Protects Endothelial Cells from Lipopolysaccharide (LPS)-Induced Inflammation by Decreasing Caspase-3 Activation and Modulating Mitochondrial Function

Acute and chronic inflammation is characterized by increased reactive oxygen species (ROS) production, dysregulation of mitochondrial metabolism and abnormal immune function contributing to cardiovascular diseases and sepsis. Clinical and epidemiological studies suggest potential beneficial effects of dietary interventions in inflammatory diseases but understanding of how nutrients work remains insufficient. In the present study, we evaluated the effects of apigenin, an anti-inflammatory flavonoid abundantly found in our diet, in endothelial cells during inflammation. Here, we show that apigenin reduced lipopolysaccharide (LPS)-induced apoptosis by decreasing ROS production and the activity of caspase-3 in endothelial cells. Apigenin conferred protection against LPS-induced mitochondrial dysfunction and reestablished normal mitochondrial complex I activity, a major site of electron leakage and superoxide production, suggesting its ability to modulate endothelial cell metabolic function during inflammation. Collectively, these findings indicate that the dietary compound apigenin stabilizes mitochondrial function during inflammation preventing endothelial cell damage and thus provide new translational opportunities for the use of dietary components in the prevention and treatment of inflammatory diseases.

A forward genetic screen identifies mutants deficient for mitochondrial complex I assembly in Chlamydomonas reinhardtii.

Mitochondrial complex I is the largest multimeric enzyme of the respiratory chain. The lack of a model system with facile genetics has limited the molecular dissection of complex I assembly. Using Chlamydomonas reinhardtii as an experimental system to screen for complex I defects, we isolated, via forward genetics, amc1–7 nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no complex I activity. Blue native (BN)-PAGE and immunoblot analyses revealed that amc3 and amc4 accumulate reduced levels of the complex I holoenzyme (950 kDa) while all other amc mutants fail to accumulate a mature complex. In amc1, -2, -5–7, the detection of a 700 kDa subcomplex retaining NADH dehydrogenase activity indicates an arrest in the assembly process. Genetic analyses established that amc5 and amc7 are alleles of the same locus while amc1–4 and amc6 define distinct complementation groups. The locus defined by the amc5 and amc7 alleles corresponds to the NUOB10 gene, encoding PDSW, a subunit of the membrane arm of complex I. This is the first report of a forward genetic screen yielding the isolation of complex I mutants. This work illustrates the potential of using Chlamydomonas as a genetically tractable organism to decipher complex I manufacture.

Transition Metal Nutrition: A Balance Between Deficiency and Toxicity

Publisher Summary This chapter describes the known components of the metal homeostasis network in Chlamydomonas, as well as the physiology and the molecular mechanisms of metal tolerance and metal deficiency responses. It delves into the metal tolerance mechanisms in Chlamydomonas and focuses on metal nutrition, deficiency, toxicity and tolerance. Unsuspected biological functions might be unveiled for these metals as more protein structures are elucidated and analytical techniques are improved. The discovery that Cd acts as the metal cofactor of a carbonic anhydrase isoform produced under Zn deficiency in the diatom Thalassiosira weissflogii suggests that metals previously considered to be non-essential and indeed toxic may occasionally act as a prosthetic group in biologically active metalloproteins. Physiological studies, genetic screens and functional analyses of candidate genes have advanced our understanding of the cellular adaptations to metal deficiency or overload in Chlamydomonas. The natural diversity of Chlamydomonas could be exploited to analyze the genetic variation associated with the metal homeostasis network within the species and genus.