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ABSTRACT: Reactive oxygen species (ROS) and autophagy have been historically associated with cell death. However, more recent evidence indicates that both ROS and autophagy play important roles in signaling and cellular adaptation to stress. As a catabolic process, autophagy allows eukaryotic cells to recycle intracellular components including entire organelles during development or under stress conditions such as nutrient limitation. Degradation and recycling of macromolecules via autophagy provides a source of building blocks (amino acids, lipids and sugars) that allow temporal adaptation of cells to adverse conditions. In addition to recycling, autophagy is required for the degradation of damaged or toxic material that can be generated as a result of ROS accumulation during oxidative stress. The mitochondrial electron-transport chain and the peroxisomes are primary sources of ROS production in most eukaryotes. The plant cell contains an additional organelle, the chloroplast, with an intense electron flow that leads to high rates of ROS production. Studies in plants and algae have demonstrated that autophagy is structurally and functionally conserved in photosynthetic organisms and plays an important role in the cellular response and adaptation to different stress conditions that involve generation of ROS such as oxidative and drought stresses, pathogen infection or photo-oxidative damage. These findings suggested a strong link between autophagy and ROS in photosynthetic eukaryotes. Here we review recent studies in plants and algae describing redox control of autophagy and discuss about conserved regulatory proteins that may transmit redox signals to the autophagic machinery.
Plant physiology 06/2012; 160(1):156-64. · 6.53 Impact Factor
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ABSTRACT: The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.
Plant physiology 08/2011; 157(2):730-41. · 6.53 Impact Factor
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ABSTRACT: Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking. Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus. The method consists of three steps: (i) enzymatic digestion in H(2)16O or isotopically enriched H(2)18O to label individual pools of differentially phosphorylated proteins; (ii) affinity selection of phosphopeptides from the combined digests by immobilized metal-affinity chromatography; and (iii) dephosphorylation with alkaline phosphatase to allow for quantitation of changes of phosphorylation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We applied this strategy to the analysis of the yeast nitrogen permease reactivator protein kinase involved in the target of rapamycin signaling pathway. Alteration in the extent of phosphorylation at Ser-353 and Ser-357 could be easily assessed and quantitated both in wild-type yeast cells treated with rapamycin and in cells lacking the SIT4 phosphatase responsible for dephosphorylating nitrogen permease reactivator protein. The method described here is simple and allows quantitation of relative changes in the level of phosphorylation in signaling proteins, thus yielding information critical for understanding the regulation of complex protein phosphorylation cascades.
Proceedings of the National Academy of Sciences 03/2003; 100(3):880-5. · 9.68 Impact Factor
