The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate-and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen-and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein.Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.

Edited by Stuart FergusonKeywords: Anaerobiosis Ferredoxin Green alga Hydrogen metabolism a b s t r a c tWe have found the transcript of one of at least six ferredoxin encoding genes of the green alga Chlamydomonas reinhardtii, FDX5, strongly accumulating in anaerobiosis, indicating a vital role of the encoded protein in the anaerobic metabolism of the cells. According to absorption and electron paramagnetic resonance spectroscopy, Fdx5 is a plant-type [2Fe2S]-ferredoxin with a redox potential similar to that of the ferredoxin PetF. However, although Fdx5 seems to be located in the chloroplast, it is not able to photoreduce nicotinamide adenine dinucleotide phosphate (NADP + ) via ferredoxin-NADP-reductase, nor to be an electron donor to the plastidic [FeFe]-hydrogenase HydA1. Thus, Fdx5 seems to have a special role in a yet to be identified anaerobic pathway.

Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. Intron-containing genes from prokaryotic or organellar genomes carry either group I or group II introns, each of which has distinct features. The splicing mechanism of group II introns and the secondary structures of their presumed active sites were used as early arguments for the hypothesis that this class of introns represents the ancestors of eukaryotic spliceosomal introns (1, 2). It was further assumed that group II introns invaded the eukaryotic nucleus and subsequently proliferated at various genomic sites, leading to the degeneration of the catalytic intron structure into small nuclear RNAs (snRNAs).1 This assumption was supported by the observation of naturally occurring variants of group II introns that are split into two or more pieces (3), reminiscent of eukaryotic spliceosomal RNA (1). Group II intron RNAs are characterized by six conserved domains, and tertiary interactions among these domains generate the compact native and catalytic complex. Some of these group II intron domains have been shown to act in trans on the splicing of other introns that lack the corresponding domain (4). In vivo, various RNA-binding proteins promote the formation of catalytically active intron RNA. In contrast to the nuclear spliceosome, which acts generally on a broad range of nuclear-encoded pre-mRNAs, proteins involved in organellar intron splicing seem to more efficiently stabilize the active three-dimensional RNA structure in vivo. Several splicing factors in higher plants, such as the chloroplast RNA-splicing and ribosome maturation (CRM) domain protein CRS1, as well as the pentatricopeptide repeat proteins OTP51 and PPR4, have been reported to be involved in the splicing of single transcripts (5, 6). Nonetheless, there are splicing factors that carry out functions on a broad range of transcripts, including CRS2 and its associated proteins CAF1 and CAF2, and WTF1, a splicing factor containing a plant organelle RNA-recognition domain (5, 6). Sedimentation and co-fractionation experiments in, for example, maize have demonstrated that these proteins are part of large multiprotein and ribonucleoprotein complexes with their cognate RNAs (5, 7). I...

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Chloroplasts are eukaryotic organelles which represent evolutionary chimera with proteins that have been derived from either a prokaryotic endosymbiont or a eukaryotic host. Chloroplast gene expression starts with transcription of RNA and is followed by multiple post-transcriptional processes which are mediated mainly by an as yet unknown number of RNA-binding proteins. Here, we review the literature to date on the structure and function of these chloroplast RNA-binding proteins. For example, the functional protein domains involved in RNA binding, such as the RNA-recognition motifs, the chloroplast RNA-splicing and ribosome maturation domains, and the pentatricopeptide-repeat motifs, are summarized. We also describe biochemical and forward genetic approaches that led to the identification of proteins modifying RNA stability or carrying out RNA splicing or editing. Such data will greatly contribute to a better understanding of the biogenesis of a unique organelle found in all photosynthetic organisms.