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.

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.In the green alga Chlamydomonas reinhardtii, the arg9-1 and arg9-2 mutations result in arginine auxotrophy because of a deficiency in N-acetyl ornithine aminotransferase activity (NAOAT) (6). Of the two arg9 mutants originally isolated (6), only the arg9-2 strain was found to be an arginine auxotroph, while the arg9-1 mutant had reverted to wild type. We reasoned that the arg9-2 mutation mapped to the structural gene for NAOAT and identified a candidate ARG9 gene (XP_ 001698091), based on the similarity of the predicted gene product to the Saccharomyces cerevisiae NAOAT, Arg8p. Three full-length cDNAs were identified and sequenced. Both the ARG9 genomic DNA and full-length cDNAs restored arginine prototrophy when introduced into the nucleus of the arg9-2 mutant (data not shown). Sequencing the ARG9 genomic locus in the arg9-2 strain identified a G-to-A transition at codon 317, resulting in a glycine-to-arginine mutation at a strictly conserved residue in NAOATs.Next, we tested if Chlamydomonas ARG9 could functionally replace the Arg8 protein in yeast by expressing the ARG9 cDNA in an arg8-null mutant that is deficient in NAOAT. Figure 1 shows that expression of the ARG9 cDNA from the plasmid-borne PGK1 promoter is able to partially restore arginine prototrophy. Since the yeast arg8 mutant can be complemented by the Chlamydomonas ARG9 protein, it is likely that the algal protein expressed in yeast is targeted to the mitochondria, where NAOAT typically functions in fungi (10). Indeed, the N-terminal extension of the candidate ARG9 protein exhibits features typical of a plastid-or mitochondriontargeting sequence, such as the propensity to form an amphiphilic ␣-helix (7).The sublocalization of the ARG9 protein was examined by immunoblot analysis using an antibody raised against Arg8p, the S. cerevisiae NAOAT that is resident in the mitochondrial matrix. The anti-Arg8p antibody cross-reacted with species in mitochondrial and plastid fractions of Chlamydomonas cells (Fig. 2). We identified the 48-kDa species in the plastid fraction as the ARG9 protein, based on the predicted size of the mature protein. This species was also present in the arg9-2 strain, suggesting that the arg9-2 mutant accumulates a nonfunctional ARG9 protein. The cross-reacting species detected in the mitochondrial fraction have higher electrophoretic mobilities and could correspond to nonspecific bands, splicing variants of the ARG9 transcript that specify a mitochondrial protein or dually targeted ARG9 protein. Complementation experiments described below indicate that the primary site of action of ARG9 is the plastid. Based on our analyses, we conc...

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In bacteria and energy generating organelles, c-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 Chlamydomonas reinhardtii through the genetic analysis of the ccs mutants (cytochrome csynthesis), which display a block in the apo- to holo- form conversion of cytochrome f and c6, the thylakoid lumen resident c-type cytochromes functioning in photosynthesis. Here we show that the gene corresponding to the CCS2 locus encodes a 1,719 amino acid polypeptide and identify the molecular lesions in the ccs2-1 to ccs2-5 alleles. The CCS2 protein displays seven degenerate amino acid repeats, which are variations of the octatricopeptide-repeat 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 Nicotiana benthamiana. We discuss the possible functions of CCS2 in the heme attachment reaction.