Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii

We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR

Molecular Advancements Establishing Chlamydomonas as a Host for Biotechnological Exploitation

peer reviewe

Production and secretion of functional SARS-CoV-2 spike protein in Chlamydomonas reinhardtii

The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1,273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to higher eukaryotes. Here we report the production of full-length SARS-CoV-2 spike protein – lacking the C-terminal membrane anchor – as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries

Fatty acid export (FAX) proteins contribute to oil production in the green microalga Chlamydomonas reinhardtii

In algae and land plants, transport of fatty acids (FAs) from their site of synthesis in the plastid stroma to the endoplasmic reticulum (ER) for assembly into acyl lipids is crucial for cellular lipid homeostasis, including the biosynthesis of triacylglycerol (TAG) for energy storage. In the unicellular green alga Chlamydomonas reinhardtii, understanding and engineering of these processes is of particular interest for microalga-based biofuel and biomaterial production. Whereas in the model plant Arabidopsis thaliana, FAX (fatty acid export) proteins have been associated with a function in plastid FA-export and hence TAG synthesis in the ER, the knowledge on the function and subcellular localization of this protein family in Chlamydomonas is still scarce. Among the four FAX proteins encoded in the Chlamydomonas genome, we found Cr-FAX1 and Cr-FAX5 to be involved in TAG production by functioning in chloroplast and ER membranes, respectively. By in situ immunolocalization, we show that Cr-FAX1 inserts into the chloroplast envelope, while Cr-FAX5 is located in ER membranes. Severe reduction of Cr-FAX1 or Cr-FAX5 proteins by an artificial microRNA approach results in a strong decrease of the TAG content in the mutant strains. Further, overexpression of chloroplast Cr-FAX1, but not of ER-intrinsic Cr-FAX5, doubled the content of TAG in Chlamydomonas cells. We therefore propose that Cr-FAX1 in chloroplast envelopes and Cr-FAX5 in ER membranes represent a basic set of FAX proteins to ensure shuttling of FAs from chloroplasts to the ER and are crucial for oil production in Chlamydomonas

Absolute Quantification of Major Photosynthetic Protein Complexes in Chlamydomonas reinhardtii Using Quantification Concatamers (QconCATs)

For modeling approaches in systems biology, knowledge of the absolute abundances of cellular proteins is essential. One way to gain this knowledge is the use of quantification concatamers (QconCATs), which are synthetic proteins consisting of proteotypic peptides derived from the target proteins to be quantified. The QconCAT protein is labeled with a heavy isotope upon expression in E. coli and known amounts of the purified protein are spiked into a whole cell protein extract. Upon tryptic digestion, labeled and unlabeled peptides are released from the QconCAT protein and the native proteins, respectively, and both are quantified by LC-MS/MS. The labeled Q-peptides then serve as standards for determining the absolute quantity of the native peptides/proteins. Here, we have applied the QconCAT approach to Chlamydomonas reinhardtii for the absolute quantification of the major proteins and protein complexes driving photosynthetic light reactions in the thylakoid membranes and carbon fixation in the pyrenoid. We found that with 25.2 attomol/cell the Rubisco large subunit makes up 6.6% of all proteins in a Chlamydomonas cell and with this exceeds the amount of the small subunit by a factor of 1.56. EPYC1, which links Rubisco to form the pyrenoid, is eight times less abundant than RBCS, and Rubisco activase is 32-times less abundant than RBCS. With 5.2 attomol/cell, photosystem II is the most abundant complex involved in the photosynthetic light reactions, followed by plastocyanin, photosystem I and the cytochrome b6/f complex, which range between 2.9 and 3.5 attomol/cell. The least abundant complex is the ATP synthase with 2 attomol/cell. While applying the QconCAT approach, we have been able to identify many potential pitfalls associated with this technique. We analyze and discuss these pitfalls in detail and provide an optimized workflow for future applications of this technique

Artificial Intelligence Understands Peptide Observability and Assists With Absolute Protein Quantification

Targeted mass spectrometry has become the method of choice to gain absolute quantification information of high quality, which is essential for a quantitative understanding of biological systems. However, the design of absolute protein quantification assays remains challenging due to variations in peptide observability and incomplete knowledge about factors influencing peptide detectability. Here, we present a deep learning algorithm for peptide detectability prediction, d::pPop, which allows the informed selection of synthetic proteotypic peptides for the successful design of targeted proteomics quantification assays. The deep neural network is able to learn a regression model that relates the physicochemical properties of a peptide to its ion intensity detected by mass spectrometry. The approach makes use of experimentally detected deviations from the assumed equimolar abundance of all peptides derived from a given protein. Trained on extensive proteomics datasets, d::pPop's plant and non-plant specific models can predict the quality of proteotypic peptides for not yet experimentally identified proteins. Interrogating the deep neural network after learning from ~76,000 peptides per model organism allows to investigate the impact of different physicochemical properties on the observability of a peptide, thus providing insights into peptide observability as a multifaceted process. Empirical evaluation with rank accuracy metrics showed that our prediction approach outperforms existing algorithms. We circumvent the delicate step of selecting positive and negative training sets and at the same time also more closely reflect the need for selecting the top most promising peptides for targeting a protein of interest. Further, we used an artificial QconCAT protein to experimentally validate the observability prediction. Our proteotypic peptide prediction approach not only facilitates the design of absolute protein quantification assays via a user-friendly web interface but also enables the selection of proteotypic peptides for not yet observed proteins, hence rendering the tool especially useful for plant research

Identification of a plastid response element that acts as an enhancer within the Chlamydomonas HSP70A promoter

Chloroplast-derived signals control a subset of nuclear genes in higher plants and eukaryotic algae. Among the types of signals identified are intermediates of chlorophyll biosynthesis such as Mg-protoporphyrin IX (MgProto). In Chlamydomonas reinhardtii, it was suggested that this tetrapyrrole mediates the light induction of chaperone gene HSP70A. Here we have analyzed cis elements involved in the regulation of HSP70A by MgProto and light. We identified two promoters and between their transcription start sites two regulatory regions that each may confer inducibility by MgProto and light to both HSP70A promoters. These regulatory regions, when cloned in front of basal non-light inducible heterologous promoters, conferred inducibility by MgProto and light. The orientation and distance independent function of these cis-regulatory sequences qualifies them as enhancers that mediate the response of nuclear genes to a chloroplast signal. Mutational analysis of one of these regulatory regions and an alignment with promoters of other MgProto-inducible genes revealed the sequence motif (G/C)CGA(C/T)N(A/G)N(15) (T/C/A)(A/T/G) which, as shown for HSP70A, may confer MgProto responsiveness. This cis-acting sequence element is employed for induction of HSP70A by both MgProto and light, lending support to the model that light induction of this gene is mediated via MgProto

VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression

VIPP proteins aid thylakoid biogenesis and membrane maintenance in cyanobacteria, algae, and plants. Some members of the Chlorophyceae contain two VIPP paralogs termed VIPP1 and VIPP2, which originate from an early gene duplication event during the evolution of green algae. VIPP2 is barely expressed under nonstress conditions but accumulates in cells exposed to high light intensities or H2O2, during recovery from heat stress, and in mutants with defective integration (alb3.1) or translocation (secA) of thylakoid membrane proteins. Recombinant VIPP2 forms rod-like structures in vitro and shows a strong affinity for phosphatidylinositol phosphate. Under stress conditions, >70% of VIPP2 is present in membrane fractions and localizes to chloroplast membranes. A vipp2 knock-out mutant displays no growth phenotypes and no defects in the biogenesis or repair of photosystem II. However, after exposure to high light intensities, the vipp2 mutant accumulates less HSP22E/F and more LHCSR3 protein and transcript. This suggests that VIPP2 modulates a retrograde signal for the expression of nuclear genes HSP22E/F and LHCSR3. Immunoprecipitation of VIPP2 from solubilized cells and membrane-enriched fractions revealed major interactions with VIPP1 and minor interactions with HSP22E/F. Our data support a distinct role of VIPP2 in sensing and coping with chloroplast membrane stress

A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP-modulated NFAT signaling

Alternative splicing is a potent modifier of protein function. Stro mal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcrip tion factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astro cytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and desta bilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteo mics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activ ity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regu late the NFAT signalosome and cAMP-SOCE crosstalk