Inhibition of Target of Rapamycin Signaling and Stress Activate Autophagy in Chlamydomonas reinhardtii

Abstract

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.

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control

“…Table 1 shows that among the up-and downregulated genes of DCH16 upon ClpP1 depletion, significant fractions, 58 and 64%, respectively, are similarly affected in the A31 strain treated with rapamycin. It should be noted, however, that the irradiance was changed from 10 to 60 mmol m 22 s 21 when rapamycin was added because induction of autophagy by rapamycin requires this light intensity (Pérez-Pérez et al, 2010). We noticed that, in some cases, this increased irradiance alone caused a change in transcript level that was larger than that induced by rapamycin The listed proteins are satisfying the following conditions: significant fold change (>2) and significant adjusted P value (<0.05).…”

“…In C. reinhardtii, Atg8 is conserved, and a recent study demonstrated that induction and lipidation of this protein can be triggered by inhibition of the TOR (target of rapamycin) protein kinase and by oxidative or endoplasmic reticulum (ER) stresses (Pérez-Pérez et al, 2010.…”

Target-of-rapamycin complex 1 (Torc1) signaling modulates cilia size and function through protein synthesis regulation

“…A plausible explanation for this incomplete blockage is the decreased potency of rapamycin in C. reinhardtii (23). Alternatively, rapamycin treatment is known to increase autophagy in the green alga (23,24), which may be able to compensate for the inhibition of translation.…”

Signaling in the plant cytosol: cysteine or sulfide?

“…Protein extracts were prepared from leaves as described (Alvarez et al 2012b ). For immunoblot analysis, 60 µg of protein extracts were resolved by 15 % SDS-PAGE and transfer to membrane that were incubated with anti-Cr-ATG8 primary (Perez-Perez et al 2010 ) and horseradish peroxidase-conjugated anti-rabbit secondary antibodies diluted 1:2,000 and 1:10,000, respectively. The previous Ponceau staining of the membrane is shown as the protein loading control.…”