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Heme attachment to the apoforms of fungal mitochondrial cytochrome c and c 1 requires the activity of cytochrome c and c 1 heme lyases (CCHL and CC 1 HL), 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 c 1 , i.e. CCHL promotes low levels of apocytochrome c 1 conversion to its holoform in the absence of CC 1 HL. 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 CC 1 HL. Multicopy and genetic suppressors of the absence of CC 1 HL were isolated and their analysis revealed that the activity of CCHL toward cytochrome c 1 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 c 1 , 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.

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.

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 b 6 f 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.

The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome. Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif. In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly. We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts. The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes. Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues. Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b 6 f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen. We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway.The c-type cytochromes are a virtually ubiquitous yet rather structurally diverse group of hemoproteins that reside on the so-called p-side 1 of the energy-transducing membrane systems where they function typically as electron carriers (1, 2). The c-type cytochromes are defined by the occurrence of heme covalently attached to the polypeptide via two thioether linkages between the vinyl groups of heme and the cysteine sulfhydryls in the apocytochrome (1, 3). A CXXCH 2 sequence in the apocytochrome provides the thiols for formation of the thioether bonds, and the imidazole of the histidine residue serves as one of the axial ligands of the heme. Remarkably, three distinct assembly pathways, referred to as systems I, II, and III, have emerged through genetic analysis of c-type cytochrome maturation in bacteria, chloroplasts, and mitochondria (for review, see Refs. 1 and 4 -7), an unexpected finding for what appears on the surface to be a rather simple chemical reaction (i.e. the addition of apocytochrome cysteine thiols to the vinyls of heme). Each system can be distinguished on the basis of a sequence relationship of specific assembly factors. Yet, there is an underlying assumption that the three systems are united by common biochemical requirements for holocytochrome c biogenesis (for review, see Refs. 2, 4, and 8). For instance, the need for reducing conditions appears to be inherent to the chemistry of thioether bond formation (4). Both substrates, apocytochrome c sulfhydryls and heme, need to be maintained reduced prior to the ligation of heme as indicated from in vitro and in o...

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.

. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions.

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-tRNA Phe . 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-tRNA Phe , and a comparable transacting activity does not exist in organelles. Although mtPheRS does not edit, it is extremely specific as only one Tyr-tRNA Phe is synthesized for every ∼7; 300 Phe-tRNA Phe , 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 wildtype 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.aminoacyl-tRNA synthetase | protein synthesis | tRNA