Patrice P. Hamel

Two Chlamydomonas CTR Copper Transporters with a Novel Cys-Met Motif Are Localized to the Plasma Membrane and Function in Copper Assimilation

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.

Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana

Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner‐membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant Δmir1 Δpic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic‐like proteins are also able to complement the double mutant Δmir1 Δpic2. 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.

Cyc2p, a Membrane-bound Flavoprotein Involved in the Maturation of Mitochondrial c-Type Cytochromes *□

Mitochondrial apocytochrome c and c1 are converted to their holoforms in the intermembrane space by attachment of heme to the cysteines of the CXXCH motif through the activity of assembly factors cytochrome c heme lyase and cytochrome c1 heme lyase (CCHL and CC1HL). The maintenance of apocytochrome sulfhydryls and heme substrates in a reduced state is critical for the ligation of heme. Factors that control the redox chemistry of the heme attachment reaction to apocytochrome c are known in bacteria and plastids but not in mitochondria. We have explored the function of Cyc2p, a candidate redox cytochrome c assembly component in yeast mitochondria. We show that Cyc2p is required for the activity of CCHL toward apocytochrome c and c1 and becomes essential for the heme attachment to apocytochrome c1 carrying a CAPCH instead of CAACH heme binding site. A redox function for Cyc2p in the heme lyase reaction is suggested from 1) the presence of a noncovalently bound FAD molecule in the C-terminal domain of Cyc2p, 2) the localization of Cyc2p in the inner membrane with the FAD binding domain exposed to the intermembrane space, and 3) the ability of recombinant Cyc2p to carry the NADPH-dependent reduction of ferricyanide. We postulate that, in vivo, Cyc2p interacts with CCHL and is involved in the reduction of heme prior to its ligation to apocytochrome c by CCHL.

Assembly of chloroplast cytochromes b and c.

The synthesis of holocytochromes in plastids is a catalyzed process. Several proteins, including plastid CcsA, Ccs1, possibly CcdA and a thioredoxin, plus at least two additional Ccs factors, are required in sub-stoichiometric amounts for the conversion of apocytochromes f and c(6) to their respective holoforms. CcsA, proposed to be a heme delivery factor, and Ccs1, an apoprotein chaperone, are speculated to interact physically in vivo. The formation of holocytochrome b(6) is a multi-step pathway in which at least four, as yet unidentified, Ccb factors are required for association of the b(H) heme. The specific requirement of reduced heme for in vitro synthesis of a cytochrome b(559)-derived holo-beta(2) suggests that cytochrome b synthesis in PSII might also be catalyzed in vivo.

Redox Processes Controlling the Biogenesis of c-Type Cytochromes

In mitochondria, two mono heme c-type cytochromes are essential electron shuttles of the respiratory chain. They are characterized by the covalent attachment of their heme C to a CXXCH motif in the apoproteins. This post-translational modification occurs in the intermembrane space compartment. Dedicated assembly pathways have evolved to achieve this chemical reaction that requires a strict reducing environment. In mitochondria, two unrelated machineries operate, the rather simple System III in yeast and animals and System I in plants and some protozoans. System I is also found in bacteria and shares some common features with System II that operates in bacteria and plastids. This review aims at presenting how different systems control the chemical requirements for the heme ligation in the compartments where cytochrome c maturation takes place. A special emphasis will be given on the redox processes that are required for the heme attachment reaction onto apocytochromes c.

The ARG9 Gene Encodes the Plastid-Resident N-Acetyl Ornithine Aminotransferase in the Green Alga Chlamydomonas reinhardtii

ABSTRACT Here we report the characterization of the Chlamydomonas reinhardtii gene ARG9, encoding the plastid resident N-acetyl ornithine aminotransferase, which is involved in arginine synthesis. Integration of an engineered ARG9 cassette in the plastid chromosome of the nuclear arg9 mutant restores arginine prototrophy. This suggests that ARG9 could be used as a new selectable marker for plastid transformation.

Role of positively charged transmembrane segments in the insertion and assembly of mitochondrial inner-membrane proteins.

The biogenesis of membrane oligomeric complexes is an intricate process that requires the insertion and assembly of transmembrane (TM) domains into the lipid bilayer. The Oxa1p family plays a key role in this process in organelles and bacteria. Hell et al. (2001, EMBO J., 20, 1281–1288) recently have proposed that Oxa1p could act as part of a general membrane insertion machinery for mitochondrial respiratory complex subunits. We have previously shown that mutations in the TM domain of Cyt1p can partially compensate for the absence of Oxa1p. Here, we demonstrate that a single amino acid substitution in the TM domain of Qcr9p can bypass Oxa1p in yeast. Qcr9p and Cyt1p are two subunits of the respiratory complex bc1 and their relative roles in the assembly of other respiratory complexes have been investigated. The mutations we have isolated in Cyt1p or Qcr9p introduce positively charged amino acids, and we show that the mutant TM domain of Cyt1p mediates the restoration of complex assembly. We propose that the positive charges introduced in Cyt1p and Qcr9p TM domains promote interactions with negatively charged TM domains of other respiratory complex subunits, allowing the coinsertion of both domains into the membrane, in the absence of Oxa1p. This model argues in favor of a role of Oxa1p in the insertion and the lateral exit of less hydrophobic TM domains from the translocation site into the lipid bilayer.

CCS5, a thioredoxin-like protein involved in the assembly of plastid c-type cytochromes

The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c6 in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b6f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c6 in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.

A Novel Component of the Disulfide-Reducing Pathway Required for Cytochrome c Assembly in Plastids

In plastids, the conversion of energy in the form of light to ATP requires key electron shuttles, the c-type cytochromes, which are defined by the covalent attachment of heme to a CXXCH motif. Plastid c-type cytochrome biogenesis occurs in the thylakoid lumen and requires a system for transmembrane transfer of reductants. Previously, CCDA and CCS5/HCF164, found in all plastid-containing organisms, have been proposed as two components of the disulfide-reducing pathway. In this work, we identify a small novel protein, CCS4, as a third component in this pathway. CCS4 was genetically identified in the green alga Chlamydomonas reinhardtii on the basis of the rescue of the ccs4 mutant, which is blocked in the synthesis of holoforms of plastid c-type cytochromes, namely cytochromes f and c6. Although CCS4 does not display sequence motifs suggestive of redox or heme-binding function, biochemical and genetic complementation experiments suggest a role in the disulfide-reducing pathway required for heme attachment to apoforms of cytochromes c. Exogenous thiols partially rescue the growth phenotype of the ccs4 mutant concomitant with recovery of holocytochrome f accumulation, as does expression of an ectopic copy of the CCDA gene, encoding a trans-thylakoid transporter of reducing equivalents. We suggest that CCS4 might function to stabilize CCDA or regulate its activity.

c-Type Cytochrome Assembly in Saccharomyces cerevisiae: A Key Residue for Apocytochrome c1/Lyase Interaction

The electron transport chains in the membranes of bacteria and organelles generate proton-motive force essential for ATP production. The c-type cytochromes, defined by the covalent attachment of heme to a CXXCH motif, are key electron carriers in these energy-transducing membranes. In mitochondria, cytochromes c and c1 are assembled by the cytochrome c heme lyases (CCHL and CC1HL) and by Cyc2p, a putative redox protein. A cytochrome c1 mutant with a CAPCH heme-binding site instead of the wild-type CAACH is strictly dependent upon Cyc2p for assembly. In this context, we found that overexpression of CC1HL, as well as mutations of the proline in the CAPCH site to H, L, S, or T residues, can bypass the absence of Cyc2p. The P mutation was postulated to shift the CXXCH motif to an oxidized form, which must be reduced in a Cyc2p-dependent reaction before heme ligation. However, measurement of the redox midpoint potential of apocytochrome c1 indicates that neither the P nor the T residues impact the thermodynamic propensity of the CXXCH motif to occur in a disulfide vs. dithiol form. We show instead that the identity of the second intervening residue in the CXXCH motif is key in determining the CCHL-dependent vs. CC1HL-dependent assembly of holocytochrome c1. We also provide evidence that Cyc2p is dedicated to the CCHL pathway and is not required for the CC1HL-dependent assembly of cytochrome c1.