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Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation.

ABSTRACT: Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.

SUBMITTER: Schreiber A

PROVIDER: S-EPMC8693860 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Multilayered Regulation of Autophagy by the Atg1 Kinase Orchestrates Spatial and Temporal Control of Autophagosome Formation

Project description:Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by utilizing de novo double membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE) stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1 dependent phosphorylation of Atg13 triggers Atg1 complex dissociation resulting in rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure. Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and inhibiting the Atg8 specific E2 and E3. Taken together, our work uncovers the molecular basis for positive and negative feedback imposed by Atg1, and opposing phosphorylation and dephosphorylation events governing the spatiotemporal regulation of autophagy.

2021-10-11 | PXD029047 | Pride

Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy.

Project description:Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. In this study, we show that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, we found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9) trafficking when cells were deprived of nutrients. Our findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein.

| S-EPMC3041946 | biostudies-literature

The autophagy-related protein kinase Atg1 interacts with the ubiquitin-like protein Atg8 via the Atg8 family interacting motif to facilitate autophagosome formation.

Project description:In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.

| S-EPMC3436563 | biostudies-literature

Atg1 kinase regulates autophagosome-vacuole fusion by controlling SNARE bundling.

Project description:Autophagy mediates the degradation of cytoplasmic material. Upon autophagy induction, autophagosomes form a sealed membrane around the cargo and fuse with the lytic compartment to release the cargo for degradation. In order to avoid premature fusion of immature autophagosomal membranes with the lytic compartment, this process needs to be tightly regulated. Several factors mediating autophagosome-vacuole fusion have recently been identified. In budding yeast, autophagosome-vacuole fusion requires the R-SNARE Ykt6 on the autophagosome, together with the three Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. However, how these SNAREs are regulated during the fusion process is poorly understood. In this study, we investigate the regulation of Ykt6. We found that Ykt6 is directly phosphorylated by Atg1 kinase, which keeps this SNARE in an inactive state. Ykt6 phosphorylation prevents SNARE bundling by disrupting its interaction with the vacuolar SNAREs Vam3 and Vti1, thereby preventing premature autophagosome-vacuole fusion. These findings shed new light on the regulation of autophagosome-vacuole fusion and reveal a further step in autophagy controlled by the Atg1 kinase.

| S-EPMC7726815 | biostudies-literature

The Atg1-kinase complex tethers Atg9-vesicles to initiate autophagy.

Project description:Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1-kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro. We find that on assembly from its cytoplasmic subcomplexes, the Atg1-kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1-kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1-Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.

| S-EPMC4729957 | biostudies-literature

ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase.

Project description:It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. Here, we have characterized the fly mutants of Autophagy-specific gene 1 (ATG1), an autophagy-regulating kinase, and found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. Our Drosophila studies have shown that ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. Consistently, overexpression of ATG1 in mammalian cells also markedly inhibits S6K in a kinase activity-dependent manner, and short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K and S6 phosphorylation. Moreover, we demonstrated that ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, our genetic and biochemical results strongly indicate crosstalk between autophagy and cell growth regulation.

| S-EPMC1852764 | biostudies-literature

An Atg1/Atg13 complex with multiple roles in TOR-mediated autophagy regulation.

Project description:The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. Here we describe a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. We show that knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1-Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling.

| S-EPMC2663935 | biostudies-literature

Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy.

Project description:Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.

| S-EPMC7883294 | biostudies-literature

Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy

Project description:Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs, however, our understanding of the mechanisms that restrain autophagy is far from complete. Here we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.

| S-SCDT-EMBOJ-2020-105120 | biostudies-other

Enhancer architecture-dependent multilayered transcriptional regulation orchestrates RA signaling-induced early lineage differentiation of ESCs.

Project description:Signaling pathway-driven target gene transcription is critical for fate determination of embryonic stem cells (ESCs), but enhancer-dependent transcriptional regulation in these processes remains poorly understood. Here, we report enhancer architecture-dependent multilayered transcriptional regulation at the Halr1-Hoxa1 locus that orchestrates retinoic acid (RA) signaling-induced early lineage differentiation of ESCs. We show that both homeobox A1 (Hoxa1) and Hoxa adjacent long non-coding RNA 1 (Halr1) are identified as direct downstream targets of RA signaling and regulated by RARA/RXRA via RA response elements (RAREs). Chromosome conformation capture-based screens indicate that RA signaling promotes enhancer interactions essential for Hoxa1 and Halr1 expression and mesendoderm differentiation of ESCs. Furthermore, the results also show that HOXA1 promotes expression of Halr1 through binding to enhancer; conversely, loss of Halr1 enhances interaction between Hoxa1 chromatin and four distal enhancers but weakens interaction with chromatin inside the HoxA cluster, leading to RA signaling-induced Hoxa1 overactivation and enhanced endoderm differentiation. These findings reveal complex transcriptional regulation involving synergistic regulation by enhancers, transcription factors and lncRNA. This work provides new insight into intrinsic molecular mechanisms underlying ESC fate determination during RA signaling-induced early differentiation.

| S-EPMC8599802 | biostudies-literature

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