Purification and characterization of protein disulphide-isomerase from the unicellular green alga Chlamydomonas reinhardii. A 120 kDa dimer antigenically distinct from the vertebrate enzyme.
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ABSTRACT:
SUBMITTER: Kaska DD
PROVIDER: S-EPMC1131391 | biostudies-other | 1990 May
REPOSITORIES: biostudies-other
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Project description:The culture medium of growing Chlamydomonas reinhardii cells contains hydroxyproline-rich glycoproteins, which are mainly liberated during release of the zoospores from the mother-cell wall. Pulse-labelling studies with [3H]proline and [35S]methionine have been performed in order to detect the protein components released by synchronously growing cells at different stages of the cell cycle. When either [3H]proline or [35S]methionine were applied during the phase of cell growth, radioactive label appeared in the released macromolecules after a lag period of 40 min, whereas incorporation into the insoluble part of the cell wall was delayed only by 20 min. When applied at the end of the growth phase, e.g. 13 h after beginning of the illumination period, the radioactive amino acids were incorporated into the cell wall, but radioactive labelling of macromolecules released into the culture medium could not be detected before the zoospores were liberated from the mother-cell wall. Maximal incorporation of [3H]proline and [35S]methionine into the insoluble part of the cell wall was observed during cell division, but essentially no radioactively-labelled macromolecules were released into the culture medium during this time period. Analysis of the macromolecules, which were liberated during cell enlargement, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed distinct radioactive bands, which were differentially labelled with [3H]proline and [35S]methionine. Among the macromolecules released into the culture medium during cell growth, a component of an apparent Mr 35 000 was preferentially labelled with [3H]proline. This component was also detected after labelling with [35S]methionine, but components of an apparently higher Mr were more prominent after labelling with [35S]methionine. Macromolecules released during the cell-enlargement period of synchronously growing cultures in the presence of [3H]proline contained radioactively-labelled hydroxyproline in addition to proline. These results show that, during cell-wall growth, specific protein components are released into the culture medium and that at least one of these components contains large amounts of proline and hydroxyproline. At least some of these macromolecules seem to be constituents of the cell wall, because during pulse-chase experiments radioactively-labelled macromolecules appeared in the culture medium mainly during the time period when the specific radioactivity of the insoluble inner-cell-wall layer decreased.
| S-EPMC1144700 | biostudies-other
Project description:In most eukaryotes, subtelomeres are dynamic genomic regions populated by multi-copy sequences of different origins, which can promote segmental duplications and chromosomal rearrangements. However, their repetitive nature has complicated the efforts to sequence them, analyse their structure and infer how they evolved. Here, we use recent genome assemblies of Chlamydomonas reinhardtii based on long-read sequencing to comprehensively describe the subtelomere architecture of the 17 chromosomes of this model unicellular green alga. We identify three main repeated elements present at subtelomeres, which we call Sultan, Subtile and Suber, alongside three chromosome extremities with ribosomal DNA as the only identified component of their subtelomeres. The most common architecture, present in 27 out of 34 subtelomeres, is a heterochromatic array of Sultan elements adjacent to the telomere, followed by a transcribed Spacer sequence, a G-rich microsatellite and transposable elements. Sequence similarity analyses suggest that Sultan elements underwent segmental duplications within each subtelomere and rearranged between subtelomeres at a much lower frequency. Analysis of other green algae reveals species-specific repeated elements that are shared across subtelomeres, with an overall organization similar to C. reinhardtii. This work uncovers the complexity and evolution of subtelomere architecture in green algae.
| S-EPMC8287924 | biostudies-literature
Project description:Endogenous small RNAs function in RNA interference (RNAi) pathways to control gene expression through mRNA cleavage, translational repression, or chromatin modification. Plants and animals contain many microRNAs (miRNAs) that play vital roles in development, including helping to specify cell type and tissue identity. To date, no miRNAs have been reported in unicellular organisms. Here we show that Chlamydomonas reinhardtii, a unicellular green alga, encodes many miRNAs. We also show that a Chlamydomonas miRNA can direct the cleavage of its target mRNA in vivo and in vitro. We further show that the expression of some miRNAs/Candidates increases or decreases during Chlamydomonas gametogenesis. In addition to miRNAs, Chlamydomonas harbors other types of small RNAs including phased small interfering RNAs (siRNAs) that are reminiscent of plant trans-acting siRNAs, as well as siRNAs originating from protein-coding genes and transposons. Our findings suggest that the miRNA pathway and some siRNA pathways are ancient mechanisms of gene regulation that evolved prior to the emergence of multicellularity.
| S-EPMC1865491 | biostudies-literature
Project description:Chlamydomonas reinhardtii is a model system for studying cilia, photosynthesis, and other core features of eukaryotes, and is also an emerging source of biofuels. Despite its importance to basic and applied biological research, the level and pattern of genetic variation in this haploid green alga has yet to be characterized on a genome-wide scale. To improve understanding of C. reinhardtii's genetic variability, we generated low coverage whole genome resequencing data for nearly all of the available isolates of this species, which were sampled from a number of sites in North America over the past ∼70 years. Based on the analysis of more than 62,000 single nucleotide polymorphisms, we identified two groups of isolates that represent geographical subpopulations of the species. We also found that measurements of genetic diversity were highly variable throughout the genome, in part due to technical factors. We studied the level and pattern of linkage disequilibrium (LD), and observed one chromosome that exhibits elevated LD. Furthermore, we detected widespread evidence of recombination across the genome, which implies that outcrossing occurs in natural populations of this species. In summary, our study provides multiple insights into the sequence diversity of C. reinhardtii that will be useful to future studies of natural genetic variation in this organism.
| S-EPMC3405113 | biostudies-literature
Project description:BackgroundPhospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction.Principal findingsWe herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC.SignificanceThis study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions.
| S-EPMC3380823 | biostudies-literature
Project description:BACKGROUND AND AIMS: Under stress-promoting conditions unicellular algae can undergo programmed cell death (PCD) but the mechanisms of algal cellular suicide are still poorly understood. In this work, the involvement of caspase-like proteases, DNA cleavage and the morphological occurrence of cell death in wasp venom mastoparan (MP)-treated Chlamydomonas reinhardtii were studied. METHODS: Algal cells were exposed to MP and cell death was analysed over time. Specific caspase inhibitors were employed to elucidate the possible role of caspase-like proteases. YVADase activity (presumably a vacuolar processing enzyme) was assayed by using a fluorogenic caspase-1 substrate. DNA breakdown was evaluated by DNA laddering and Comet analysis. Cellular morphology was examined by confocal laser scanning microscopy. KEY RESULTS: MP-treated C. reinhardtii cells expressed several features of necrosis (protoplast shrinkage) and vacuolar cell death (lytic vesicles, vacuolization, empty cell-walled corpse-containing remains of digested protoplast) sometimes within one single cell and in different individual cells. Nucleus compaction and DNA fragmentation were detected. YVADase activity was rapidly stimulated in response to MP but the early cell death was not inhibited by caspase inhibitors. At later time points, however, the caspase inhibitors were effective in cell-death suppression. Conditioned medium from MP-treated cells offered protection against MP-induced cell death. CONCLUSIONS: In C. reinhardtii MP triggered PCD of atypical phenotype comprising features of vacuolar and necrotic cell deaths, reminiscent of the modality of hypersensitive response. It was assumed that depending on the physiological state and sensitivity of the cells to MP, the early cell-death phase might be not mediated by caspase-like enzymes, whereas later cell death may involve caspase-like-dependent proteolysis. The findings substantiate the hypothesis that, depending on the mode of induction and sensitivity of the cells, algal PCD may take different forms and proceed through different pathways.
| S-EPMC3555528 | biostudies-other
Project description:Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO2 retro-diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed algal SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing 'pyrenoid-competent' chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild-type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.
| S-EPMC5363358 | biostudies-literature
Project description:Capturing a valid snapshot of the metabolome requires rapid quenching of enzyme activities. This is a crucial step in order to halt the constant flux of metabolism and high turnover rate of metabolites. Quenching with cold aqueous methanol is treated as a gold standard so far, however, reliability of metabolomics data obtained is in question due to potential problems connected to leakage of intracellular metabolites. Therefore, we investigated the influence of various parameters such as quenching solvents, methanol concentration, inclusion of buffer additives, quenching time and solvent to sample ratio on intracellular metabolite leakage from Chlamydomonas reinhardtii. We measured the recovery of twelve metabolite classes using gas chromatography mass spectrometry (GC-MS) in all possible fractions and established mass balance to trace the fate of metabolites during quenching treatments. Our data demonstrate significant loss of intracellular metabolites with the use of the conventional 60% methanol, and that an increase in methanol concentration or quenching time also resulted in higher leakage. Inclusion of various buffer additives showed 70 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) to be suitable. In summary, we recommend quenching with 60% aqueous methanol supplemented with 70 mM HEPES (-40 °C) at 1:1 sample to quenching solvent ratio, as it resulted in higher recoveries for intracellular metabolites with subsequent reduction in the metabolite leakage for all metabolite classes.
| S-EPMC6315863 | biostudies-literature
Project description:The molecular function of mTERFs (mitochondrial transcription termination factors) has so far only been described for metazoan members of the protein family and in animals they control mitochondrial replication, transcription and translation. Cells of photosynthetic eukaryotes harbour chloroplasts and mitochondria, which are in an intense cross-talk that is vital for photosynthesis. Chlamydomonas reinhardtii is a unicellular green alga widely used as a model organism for photosynthesis research and green biotechnology. Among the six nuclear C. reinhardtii mTERF genes is mTERF-like gene of Chlamydomonas (MOC1), whose inactivation alters mitorespiration and interestingly also light-acclimation processes in the chloroplast that favour the enhanced production of biohydrogen. We show here from in vitro studies that MOC1 binds specifically to a sequence within the mitochondrial rRNA-coding module S3, and that a knockout of MOC1 in the mutant stm6 increases read-through transcription at this site, indicating that MOC1 acts as a transcription terminator in vivo. Whereas the level of certain antisense RNA species is higher in stm6, the amount of unprocessed mitochondrial sense transcripts is strongly reduced, demonstrating that a loss of MOC1 causes perturbed mitochondrial DNA (mtDNA) expression. Overall, we provide evidence for the existence of mitochondrial antisense RNAs in C. reinhardtii and show that mTERF-mediated transcription termination is an evolutionary-conserved mechanism occurring in phototrophic protists and metazoans.
| S-EPMC3711419 | biostudies-literature
Project description:Comparative genomic analysis of a photosymbiont evolution
| PRJNA832740 | ENA