Dataset Information

Activation of Autophagy by Metals in Chlamydomonas reinhardtii.

ABSTRACT: Autophagy is an intracellular self-degradation pathway by which eukaryotic cells recycle their own material in response to specific stress conditions. Exposure to high concentrations of metals causes cell damage, although the effect of metal stress on autophagy has not been explored in photosynthetic organisms. In this study, we investigated the effect of metal excess on autophagy in the model unicellular green alga Chlamydomonas reinhardtii. We show in cells treated with nickel an upregulation of ATG8 that is independent of CRR1, a global regulator of copper signaling in Chlamydomonas. A similar effect on ATG8 was observed with copper and cobalt but not with cadmium or mercury ions. Transcriptome sequencing data revealed an increase in the abundance of the protein degradation machinery, including that responsible for autophagy, and a substantial overlap of that increased abundance with the hydrogen peroxide response in cells treated with nickel ions. Thus, our results indicate that metal stress triggers autophagy in Chlamydomonas and suggest that excess nickel may cause oxidative damage, which in turn activates degradative pathways, including autophagy, to clear impaired components and recover cellular homeostasis.

SUBMITTER: Perez-Martin M

PROVIDER: S-EPMC4551596 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Activation of Autophagy by Metals in Chlamydomonas reinhardtii.

Pérez-Martín Marta M Blaby-Haas Crysten E CE Pérez-Pérez María Esther ME Andrés-Garrido Ascensión A Blaby Ian K IK Merchant Sabeeha S SS Crespo José L JL

Eukaryotic cell 20150710 9

Autophagy is an intracellular self-degradation pathway by which eukaryotic cells recycle their own material in response to specific stress conditions. Exposure to high concentrations of metals causes cell damage, although the effect of metal stress on autophagy has not been explored in photosynthetic organisms. In this study, we investigated the effect of metal excess on autophagy in the model unicellular green alga Chlamydomonas reinhardtii. We show in cells treated with nickel an upregulation ...[more]

PMID: 26163317

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Project description:Autophagy plays a role in regulating important cellular functions in response to stress conditions. The role of nitric oxide (NO) in the regulation of autophagy in Chlamydomonas reinhardtii has been not studied. Illumination of C. reinhardtii cells under a high light (HL, 1,600 ?mol m-2 s-1) condition induced a NO burst through NO synthase- and nitrate reductase-independent routes, and cell death. The abundance of CrATG8 protein, an autophagy marker of C. reinhardtii, increased after HL illumination along with a linear increase in the transcript abundance of autophagy-associated genes (CrVPS34, CrATG1, CrATG3, CrATG4, CrATG6, CrATG7, CrATG8, and CrATG12), which were suppressed in the presence of an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). The cells were treated with NO donors, S-nitroso-N-acetyl-penicillamine, and S-nitrosoglutathione, under a normal light (50 ?mol m-2 s-1) condition to elucidate the role of NO in autophagy activation and cell death. Treatment with 0.05 mM or 0.1 mM NO donors increased the abundance of ATG8 protein and CrATG transcripts, which were suppressed in the presence of cPTIO. Moreover, treatment with 0.05 mM NO donors did not affect cell viability, while 0.1 mM NO donors elicited a transient decrease in cell growth and death that recovered after 12 h. The transient effect could be prevented by the presence of cPTIO. However, treatment with 1 mM H2O2 and 0.1 mM NO donors enhanced autophagy induction and resulted in cell death after 24 h. The interaction of H2O2 and NO can be prevented by cPTIO treatment. This implies that NO is critical for the interaction of H2O2 and NO that induces cell death and autophagy. Furthermore, exposure to 0.1 mM NO donors under a non-lethal HL condition (750 ?mol m-2 s-1) evoked autophagy and cell death. In conclusion, the present findings demonstrated that the NO-mediated autophagy pathway is activated in C. reinhardtii under lethal high intensity illumination and may interact with H2O2 for HL-induced cell death. The relationships between autophagy and cell death are discussed.

Project description:BackgroundCah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2) fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2) conditions.Results/conclusionsIn the present work we demonstrate that after transfer to low CO(2), Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO(2) conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO(2) conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO(2) conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO(2) the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules.SignificanceThis is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Dataset Information

Activation of Autophagy by Metals in Chlamydomonas reinhardtii.

Publications

Activation of Autophagy by Metals in Chlamydomonas reinhardtii.

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