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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 Ito1 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.

Cytochromes are hemoproteins, with the prosthetic group covalently linked to the apoprotein, which function as electron carriers. A class of cytochromes is defined by a C CH heme-binding motif where the cysteines form thioether bonds with the vinyl groups of heme. Plastids are known to contain up to three cytochromes . The membrane-bound cytochrome and soluble cytochrome operate in photosynthesis while the activity of soluble cytochrome remains unknown. Conversion of apo- to holocytochrome occurs in the thylakoid lumen and requires the independent transport of apocytochrome and heme across the thylakoid membrane followed by the stereospecific attachment of ferroheme via thioether linkages. Attachment of heme to apoforms of plastid cytochromes is dependent upon the products of the (for ytochrome ynthesis) genes, first uncovered via genetic analysis of photosynthetic deficient mutants in the green alga . The CCS pathway also occurs in cyanobacteria and several bacteria. CcsA and CCS1, the signature components of the CCS pathway are polytopic membrane proteins proposed to operate in the delivery of heme from the stroma to the lumen, and also in the catalysis of the heme ligation reaction. CCDA, CCS4, and CCS5 are components of trans-thylakoid pathways that deliver reducing equivalents in order to maintain the heme-binding cysteines in a reduced form prior to thioether bond formation. While only four CCS components are needed in bacteria, at least eight components are required for plastid cytochrome assembly, suggesting the biochemistry of thioether formation is more nuanced in the plastid system.

Inducible high-affinity copper uptake is key to copper homeostasis in Chlamydomonas reinhardtii. We generated cDNAs and updated gene models for four genes, CTR1, CTR2, CTR3, and COPT1, encoding CTR-type copper transporters in CHLAMYDOMONAS: The expression of CTR1, CTR2, and CTR3 increases in copper deficient cells and in response to hypoxia or Ni²⁺ supplementation; this response depends on the transcriptional activator CRR1. A copper response element was identified by mutational analysis of the 5' upstream region of CTR1. Functional analyses identify CTR1 and CTR2 as the assimilatory transporters of Chlamydomonas based on localization to the plasma membrane and ability to rescue a Saccharomyces cerevisiae mutant defective in high-affinity copper transport. The Chlamydomonas CTRs contain a novel Cys-Met motif (CxxMxxMxxC-x₅/₆-C), which occurs also in homologous proteins in other green algae, amoebae, and pathogenic fungi. CTR3 appears to have arisen by duplication of CTR2, but CTR3 lacks the characteristic transmembrane domains found in the transporters, suggesting that it may be a soluble protein. Thus, Chlamydomonas CTR genes encode a distinct subset of the classical CTR family of Cu(I) transporters and represent new targets of CRR1-dependent signaling.

Allotopic expression refers to the artificial relocation of an organellar gene to the nucleus. Subunit 2 (Cox2) of cytochrome c oxidase, a subunit with 2 transmembrane domains (TMS1 and TMS2) residing in the inner mitochondrial membrane with a Nout-Cout topology, is typically encoded in the mitochondrial cox2 gene. In the yeast Saccharomyces cerevisiae, the cox2 gene can be allotopically expressed in the nucleus, yielding a functional protein that restores respiratory growth to a Δcox2 null mutant. In addition to a mitochondrial targeting sequence followed by its natural 15-residue leader peptide, the cytosol synthesized Cox2 precursor must carry one or several amino acid substitutions that decrease the mean hydrophobicity of TMS1 and facilitate its import into the matrix by the TIM23 translocase. Here, using a yeast strain that contains a COX2W56R gene construct inserted in a nuclear chromosome, we searched for genes whose overexpression could facilitate import into mitochondria of the Cox2W56R precursor and increase respiratory growth of the corresponding mutant strain. A COX2W56R expressing strain was transformed with a multicopy plasmid genomic library, and transformants exhibiting enhanced respiratory growth on nonfermentable carbon sources were selected. We identified 3 genes whose overexpression facilitates the internalization of the Cox2W56R subunit into mitochondria, namely: TYE7, RAS2, and COX12. TYE7 encodes a transcriptional factor, RAS2, a GTP-binding protein, and COX12, a non-core subunit of cytochrome c oxidase. We discuss potential mechanisms by which the TYE7, RAS2, and COX12 gene products could facilitate the import and assembly of the Cox2W56R subunit produced allotopically.

In bacteria and energy generating organelles, -type cytochromes are a class of universal electron carriers with a heme cofactor covalently linked via one or two thioether bonds to a heme binding site. The covalent attachment of heme to apocytochromes is a catalyzed process, taking place via three evolutionarily distinct assembly pathways (Systems I, II, III). System II was discovered in the green alga through the genetic analysis of the mutants ( ytochrome ynthesis), which display a block in the apo- to holo- form conversion of cytochrome and , the thylakoid lumen resident -type cytochromes functioning in photosynthesis. Here we show that the gene corresponding to the locus encodes a 1,719 amino acid polypeptide and identify the molecular lesions in the to alleles. The CCS2 protein displays seven degenerate amino acid repeats, which are variations of the ctatrico eptide- epeat motif (OPR) recently recognized in several nuclear-encoded proteins controlling the maturation, stability, or translation of chloroplast transcripts. A plastid site of action for CCS2 is inferred from the finding that GFP fused to the first 100 amino acids of the algal protein localizes to chloroplasts in . We discuss the possible functions of CCS2 in the heme attachment reaction.

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.

Summary Fungal infections, a leading cause of mortality among eukaryotic pathogens, pose a growing global health threat due to the rise of drug-resistant strains. New therapeutic strategies are urgently needed to combat this challenge. The PCA pathway for biosynthesis of Co-enzyme A (CoA) and Acetyl-CoA (AcCoA) from vitamin B5 (pantothenic acid) has been validated as an excellent target for the development of new antimicrobials against fungi and protozoa. The pathway regulates key cellular processes including metabolism of fatty acids, amino acids, sterols, and heme. In this study, we provide genetic evidence that disruption of the PCA pathway in Saccharomyces cerevisiae results in a significant alteration in the susceptibility of fungi to a wide range of xenobiotics, including clinically approved antifungal drugs through alteration of vacuolar morphology and drug detoxification. The drug potentiation mediated by genetic regulation of genes in the PCA pathway could be recapitulated using the pantazine analog PZ-2891 as well as the celecoxib derivative, AR-12 through inhibition of fungal AcCoA synthase activity. Collectively, the data validate the PCA pathway as a suitable target for enhancing the efficacy and safety of current antifungal therapies. The PCA pathway, essential for synthesizing CoA and AcCoA from vitamin B5, plays a crucial role in fungal cellular processes. Targeting this pathway could enhance antifungal drug efficacy by affecting vacuolar morphology and drug detoxification.

Thiol oxidation to disulfides and the reverse reaction, i.e., disulfide reduction to free thiols, are under the control of catalysts in vivo. Enzymatically assisted thiol-disulfide chemistry is required for the biogenesis of all energy-transducing membrane systems. However, until recently, this had only been demonstrated for the bacterial plasma membrane. Long considered to be vacant, the thylakoid lumen has now moved to the forefront of photosynthesis research with the realization that its proteome is far more complicated than initially anticipated. Several lumenal proteins are known to be disulfide bonded in Arabidopsis, highlighting the importance of sulfhydryl oxidation in the thylakoid lumen. While disulfide reduction in the plastid stroma is known to activate several enzymatic activities, it appears that it is the reverse reaction, i.e., thiol oxidation that is required for the activity of several lumen-resident proteins. This paradigm for redox regulation in the thylakoid lumen has opened a new frontier for research in the field of photosynthesis. Of particular significance in this context is the discovery of trans-thylakoid redox pathways controlling disulfide bond formation and reduction, which are required for photosynthesis.

With 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 complex I mitochondrial genes in this organism.

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.