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Autophagy is a central degradative pathway highly conserved among eukaryotes, including microalgae, which remains unexplored in extremophilic organisms. In this study, we described and characterized autophagy in the newly identified extremophilic green microalga Chlamydomonas urium, which was isolated from an acidic environment. The nuclear genome of C. urium was sequenced, assembled and annotated in order to identify autophagy-related genes. Transmission electron microscopy, immunoblotting, metabolomic and photosynthetic analyses were performed to investigate autophagy in this extremophilic microalga. The analysis of the C. urium genome revealed the conservation of core autophagy-related genes. We investigated the role of autophagy in C. urium by blocking autophagic flux with the vacuolar ATPase inhibitor concanamycin A. Our results indicated that inhibition of autophagic flux in this microalga resulted in a pronounced accumulation of triacylglycerols and lipid droplets (LDs). Metabolomic and photosynthetic analyses indicated that C. urium cells with impaired vacuolar function maintained an active metabolism. Such effects were not observed in the neutrophilic microalga Chlamydomonas reinhardtii. Inhibition of autophagic flux in C. urium uncovered an active recycling of LDs through lipophagy, a selective autophagy pathway for lipid turnover. This study provided the metabolic basis by which extremophilic algae are able to catabolize lipids in the vacuole., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Autophagy is a catabolic process by which eukaryotic cells degrade and recycle unnecessary or damaged intracellular components to maintain cellular homeostasis and to cope with stress. The development of specific tools to monitor autophagy in microalgae and plants has been fundamental to investigate this catabolic pathway in photosynthetic organisms. The protein ATG8 is a widely used molecular marker of autophagy in all eukaryotes, including the model microalga Chlamydomonas reinhardtii. The drug concanamycin A, a specific inhibitor of vacuolar ATPase, has also been extensively used to block autophagic flux in the green lineage. In Chlamydomonas, inhibition of autophagic flux by concanamycin A has been shown to prevent the degradation of ribosomal proteins and the formation of lipid bodies under nitrogen or phosphorous starvation. Here, we detail how the abundance and lipidation state of ATG8 can be used to monitor autophagic flux in Chlamydomonas by western blot analysis., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Deciphering the molecular mechanisms underlying the regulation of the ATG4 protease is essential to understand the regulation of ATG8 lipidation, a key step in the biogenesis of the autophagosome and hence in autophagy progression. Here, we describe two complementary approaches to monitor ATG4 proteolytic activity in the model green alga Chlamydomonas reinhardtii: an in vitro assay using recombinant ATG4 and recombinant ATG8 as substrate, and a cell-free assay using soluble total protein extract from Chlamydomonas and recombinant Chlamydomonas ATG8 as substrate. Both assays are followed by non-reducing SDS-PAGE and immuno-blot analysis. Given the high evolutionary conservation of the ATG8 maturation process, these assays have also been validated to monitor ATG4 activity in yeast using Chlamydomonas ATG8 as substrate., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Stress and nutrient availability influence cell proliferation through complex intracellular signalling networks. In a previous study it was found that pyro-inositol polyphosphates (InsP 7 and InsP 8 ) produced by VIP1 kinase, and target of rapamycin (TOR) kinase signalling interacted synergistically to control cell growth and lipid metabolism in the green alga Chlamydomonas reinhardtii. However, the relationship between InsPs and TOR was not completely elucidated. We used an in vivo assay for TOR activity together with global proteomic and phosphoproteomic analyses to assess differences between wild-type and vip1-1 in the presence and absence of rapamycin. We found that TOR signalling is more severely affected by the inhibitor rapamycin in a vip1-1 mutant compared with wild-type, indicating that InsP 7 and InsP 8 produced by VIP1 act independently but also coordinately with TOR. Additionally, among hundreds of differentially phosphorylated peptides detected, an enrichment for photosynthesis-related proteins was observed, particularly photosystem II proteins. The significance of these results was underscored by the finding that vip1-1 strains show multiple defects in photosynthetic physiology that were exacerbated under high light conditions. These results suggest a novel role for inositol pyrophosphates and TOR signalling in coordinating photosystem phosphorylation patterns in Chlamydomonas cells in response to light stress and possibly other stresses., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas ( Chlamydomonas reinhardtii ) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas., (© 2020 American Society of Plant Biologists. All rights reserved.)

Autophagy is a major catabolic pathway by which eukaryotic cells deliver unnecessary or damaged cytoplasmic material to the vacuole for its degradation and recycling in order to maintain cellular homeostasis. Control of autophagy has been associated with the production of reactive oxygen species in several organisms, including plants and algae, but the precise regulatory molecular mechanisms remain unclear. Here, we show that the ATG4 protease, an essential protein for autophagosome biogenesis, plays a central role for the redox regulation of autophagy in the model green alga Chlamydomonas reinhardtii Our results indicate that the activity of C. reinhardtii ATG4 is regulated by the formation of a single disulfide bond with a low redox potential that can be efficiently reduced by the NADPH/thioredoxin system. Moreover, we found that treatment of C. reinhardtii cells with norflurazon, an inhibitor of carotenoid biosynthesis that generates reactive oxygen species and triggers autophagy in this alga, promotes the oxidation and aggregation of ATG4. We propose that the activity of the ATG4 protease is finely regulated by the intracellular redox state, and it is inhibited under stress conditions to ensure lipidation of ATG8 and thus autophagy progression in C. reinhardtii., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Microalgae constitute a highly diverse group of photosynthetic microorganisms that are widely distributed on Earth. The rich diversity of microalgae arose from endosymbiotic events that took place early in the evolution of eukaryotes and gave rise to multiple lineages including green algae, the ancestors of land plants. In addition to their fundamental role as the primary source of marine and freshwater food chains, microalgae are essential producers of oxygen on the planet and a major biotechnological target for sustainable biofuel production and CO2 mitigation. Microalgae integrate light and nutrient signals to regulate cell growth. Recent studies identified the target of rapamycin (TOR) kinase as a central regulator of cell growth and a nutrient sensor in microalgae. TOR promotes protein synthesis and regulates processes that are induced under nutrient stress such as autophagy and the accumulation of triacylglycerol and starch. A detailed analysis of representative genomes from the entire microalgal lineage revealed that the highly conserved central components of the TOR pathway are likely to have been present in the last eukaryotic common ancestor, and the loss of specific TOR signaling elements at an early stage in the evolution of microalgae. Here we examine the evolutionary conservation of TOR signaling components in diverse microalgae and discuss recent progress of this signaling pathway in these organisms. [ABSTRACT FROM AUTHOR]

The target of rapamycin (TOR) kinase is a master regulator that integrates nutrient signals to promote cell growth in all eukaryotes. It is well established that amino acids and glucose are major regulators of TOR signaling in yeast and metazoan, but whether and how TOR responds to carbon availability in photosynthetic organisms is less understood. In this study, we showed that photosynthetic assimilation of CO2 by the Calvin–Benson–Bassham (CBB) cycle regulates TOR activity in the model single-celled microalga Chlamydomonas reinhardtii. Stimulation of CO2 fixation boosted TOR activity, whereas inhibition of the CBB cycle and photosynthesis down-regulated TOR. We uncovered a tight link between TOR activity and the endogenous level of a set of amino acids including Ala, Glu, Gln, Leu, and Val through the modulation of CO2 fixation and the use of amino acid synthesis inhibitors. Moreover, the finding that the Chlamydomonas starch-deficient mutant sta6 displayed disproportionate TOR activity and high levels of most amino acids, particularly Gln, further connected carbon assimilation and amino acids to TOR signaling. Thus, our results showed that CO2 fixation regulates TOR signaling, likely through the synthesis of key amino acids. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved degradative pathway that ensures cellular homeostasis through the removal of damaged or useless intracellular components including proteins, membranes, or even entire organelles. A main hallmark of autophagy is the biogenesis of autophagosomes, double-membrane vesicles that engulf and transport to the vacuole the material to be degraded and recycled. The formation of autophagosomes responds to integrated signals produced as a consequence of metabolic reactions or different types of stress and is mediated by the coordinated action of core autophagy-related (ATG) proteins. ATG4 is a key Cys-protease with a dual function in both ATG8 lipidation and free ATG8 recycling whose balance is crucial for proper biogenesis of the autophagosome. ATG4 is conserved in the green lineage, and its regulation by different post-translational modifications has been reported in the model systems Chlamydomonas reinhardtii and Arabidopsis. In this review, we discuss the major role of ATG4 in the integration of stress and redox signals that regulate autophagy in algae and plants. [ABSTRACT FROM AUTHOR]

Autophagy is an intracellular catabolic system that delivers cytoplasmic constituents and organelles in the vacuole. This degradative process is mediated by a group of proteins coded by autophagy-related (ATG) genes that are widely conserved from yeasts to plants and mammals. Homologs of ATG genes have been also identified in algal genomes including the unicellular model green alga Chlamydomonas reinhardtii. The development of specific tools to monitor autophagy in Chlamydomonas has expanded our current knowledge about the regulation and function of this process in algae. Recent findings indicated that autophagy is regulated by redox signals and the TOR network in Chlamydomonas and revealed that this process may play in important role in the control of lipid metabolism and ribosomal protein turnover in this alga. Here, we will describe the different techniques and approaches that have been reported to study autophagy and autophagic flux in Chlamydomonas. [ABSTRACT FROM AUTHOR]

Cell growth is tightly coupled to nutrient availability. The target of rapamycin (TOR) kinase transmits nutritional and environmental cues to the cellular growth machinery. TOR functions in two distinct multiprotein complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). While the structure and functions of TORC1 are highly conserved in all eukaryotes, including algae and plants, TORC2 core proteins seem to be missing in photosynthetic organisms. TORC1 controls cell growth by promoting anabolic processes, including protein synthesis and ribosome biogenesis, and inhibiting catabolic processes such as autophagy. Recent studies identified rapamycin-sensitive TORC1 signaling regulating cell growth, autophagy, lipid metabolism, and central metabolic pathways in the model unicellular green alga Chlamydomonas reinhardtii. The central role that microalgae play in global biomass production, together with the high biotechnological potential of these organisms in biofuel production, has drawn attention to the study of proteins that regulate cell growth such as the TOR kinase. In this review we discuss the recent progress on TOR signaling in algae. [ABSTRACT FROM AUTHOR]

Autophagy is a catabolic membrane-trafficking process whereby cells recycle cytosolic proteins and organelles under stress conditions or during development. This degradative process is mediated by autophagy-related (ATG) proteins that have been described in yeast, animals, and more recently in plants. In this study, we report the molecular characterization of autophagy in the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) is functionally conserved and may be used as a molecular autophagy marker. Like yeast ATG8, CrATG8 is cleaved at the carboxyl-terminal conserved glycine and is associated with membranes in Chlamydomonas. Cell aging or different stresses such as nutrient limitation, oxidative stress, or the accumulation of misfolded proteins in the endoplasmic reticulum caused an increase in CrATG8 abundance as well as the detection of modified forms of this protein, both landmarks of autophagy activation. Furthermore, rapamycin-mediated inhibition of the Target of Rapamycin signaling pathway, a major regulator of autophagy in eukaryotes, results in identical effects on CrATG8 and a relocalization of this protein in Chlamydomonas cells similar to the one observed upon nutrient limitation. Thus, our findings indicate that Chlamydomonas cells may respond to stress conditions by inducing autophagy via Target of Rapamycin signaling modulation. [ABSTRACT FROM AUTHOR]

The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved catabolic process in eukaryotic cells by which waste cellular components are recycled to maintain growth in both favorable and stress conditions. Autophagy has been linked to lipid metabolism in microalgae; however, the mechanism underlying this interaction remains unclear. In this study, transgenic Chlamydomonas reinhardtii cells that stably express the red fluorescent protein (mCherry) tagged-ATG8 as an autophagy marker were established. By using this tool, we were able to follow the autophagy process in live microalgal cells under various conditions. Live-cell and transmission electron microscopy (TEM) imaging revealed physical contacts between lipid droplets and autophagic structures during the early stage of nitrogen starvation, while fusion of these two organelles was observed in prolonged nutritional deficiency, suggesting that an autophagy-related pathway might be involved in lipid droplet turnover in this alga. Our results thus shed light on the interplay between autophagy and lipid metabolism in C. reinhardtii, and this autophagy marker would be a valuable asset for further investigations on autophagic processes in microalgae. [ABSTRACT FROM AUTHOR]