ABSTRACT
Target of rapamycin complex 1 (TORC1) protein kinase responds to various stresses including genotoxic stress. However, its molecular mechanism is poorly understood. Here, we show that DNA damage induces nonselective and selective autophagy in budding yeast. DNA damage caused the attenuation of TORC1 activity, dephosphorylation of Atg13, and autophagy induction. The TORC1-upstream Rag GTPase Gtr1 was not required for TORC1 inactivation and autophagy induction after DNA damage. Furthermore, DNA damage responsive protein kinases Mec1/ATM and Tel1/ATR, and stress-responsive mitogen-activated protein kinase Mpk1/Slt2 were required for the full induction of autophagy. Autophagic proteolysis was required for DNA damage tolerance in TORC1 inactive conditions. This study revealed that multiple protein kinases regulate DNA damage-induced autophagy.
Subject(s)
Autophagy/genetics , Intracellular Signaling Peptides and Proteins/genetics , Mitogen-Activated Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , Adaptor Proteins, Signal Transducing/genetics , Autophagy-Related Proteins/genetics , DNA Damage/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Fungal/genetics , Mechanistic Target of Rapamycin Complex 1/genetics , Monomeric GTP-Binding Proteins/genetics , Saccharomyces cerevisiae/genetics , Signal Transduction/geneticsABSTRACT
Misfolded and aggregated proteins are eliminated to maintain protein homeostasis. Autophagy contributes to the removal of protein aggregates. However, if and how proteotoxic stress induces autophagy is poorly understood. Here we show that proteotoxic stress after treatment with azetidine-2-carboxylic acid (AZC), a toxic proline analog, induces autophagy in budding yeast. AZC treatment attenuated target of rapamycin complex 1 (TORC1) activity, resulting in the dephosphorylation of Atg13, a key factor of autophagy. By contrast, AZC treatment did not affect target of rapamycin complex 2 (TORC2). Proteotoxic stress also induced TORC1 inactivation and autophagy in fission yeast and human cells. This study suggested that TORC1 is a conserved key factor to cope with proteotoxic stress in eukaryotic cells.
Subject(s)
Autophagy/drug effects , Azetidinecarboxylic Acid/toxicity , Mechanistic Target of Rapamycin Complex 1/metabolism , Saccharomyces cerevisiae/drug effects , Schizosaccharomyces/drug effects , Fungal Proteins/metabolism , HEK293 Cells , Humans , Saccharomyces cerevisiae/cytology , Schizosaccharomyces/cytologyABSTRACT
Target of rapamycin complex 1 (TORC1) protein kinase, a master controller of cell growth, is thought to be involved in genome integrity. However, the molecular mechanisms associated with this are unclear. Here, we show that TORC1 inactivation causes decreases in the levels of a wide range of proteins involved in the DNA damage checkpoint (DDC) signaling including Tel1, Mre11, Rad9, Mrc1, and Chk1 in budding yeast. Furthermore, TORC1 inactivation compromised DDC activation, DNA repair, and cell survival after DNA damage. TORC1 inactivation promoted proteasomal degradation of Rad9 and Mre11 in a manner dependent on Skp1-Cullin-F-box protein (SCF). Finally, CDK promoted the degradation of Rad9. This study revealed that TORC1 is essential for genome integrity via the maintenance of DDC signaling.
Subject(s)
DNA Damage , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Cell Cycle Proteins/metabolism , DNA Repair , DNA Replication , Endodeoxyribonucleases/metabolism , Exodeoxyribonucleases/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteolysis , Saccharomyces cerevisiae/geneticsABSTRACT
Autophagic degradation of ribosomes is promoted by nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1). Here we show that selective autophagic degradation of ribosomes (called ribophagy) after TORC1 inactivation requires the specific autophagy receptor Atg11. Rim15 protein kinase upregulated ribophagy, while it downregulated non-selective degradation of ribosomes.
Subject(s)
Autophagy , Protein Kinases/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Autophagy-Related Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
Target of rapamycin complex 1 (TORC1) phosphorylates autophagy-related Atg13 and represses autophagy under nutrient-rich conditions. However, when TORC1 becomes inactive upon nutrient depletion or treatment with the TORC1 inhibitor rapamycin, Atg13 dephosphorylation occurs rapidly, and autophagy is induced. At present, the phosphatases involved in Atg13 dephosphorylation remain unknown. Here, we show that two protein phosphatase 2A (PP2A) phosphatases, PP2A-Cdc55 and PP2A-Rts1, which are activated by inactivation of TORC1, are required for sufficient Atg13 dephosphorylation and autophagy induction after TORC1 inactivation in budding yeast. After rapamycin treatment, dephosphorylation of Atg13, activation of Atg1 kinase, pre-autophagosomal structure (PAS) formation and autophagy induction are all impaired in PP2A-deleted cells. Conversely, overexpression of non-phosphorylatable Atg13 suppressed defects in autophagy in PP2A mutant. This study revealed that the orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1.