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There are 10 results for ATG11 (displaying 1 to 10).

Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11

The selective autophagy receptors Atg19 and Atg32 interact with two proteins of the core autophagic machinery: the scaffold protein Atg11 and the ubiquitin‐like protein Atg8. We found that the Pichia pastoris pexophagy receptor, Atg30, also interacts with Atg8. Both Atg30 and Atg32 interactions are regulated by phosphorylation close to Atg8‐interaction motifs. Extending this finding to Saccharomyces cerevisiae , we confirmed phosphoregulation for the mitophagy and pexophagy receptors, Atg32 …

Interactions between Atg30, Atg8 and Atg11. (A1) Atg30 sequences from aa 68–122 of wild‐type (Atg30), the S71A mutant (Atg30S71A) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 (Atg30WDILSSS and Atg30WSILSSS). (A2) Pexophagy experiments of Δatg30 cells complemented with appropriate wild‐type or mutant Atg30 proteins described in A1. (A3) Two‐hybrid assays between Atg30 wild‐type or mutants (described in A1) and Atg8. Phosphomimic …

Atg8 and Atg11 localization during pexophagy. (A) Large phagophore membrane formation in WT and the Atg30 mutant cells monitored by GFP–Atg8 during pexophagy conditions. (B) Localization of GFP–Atg11 during pexophagy of methanol‐induced peroxisomes in cells expressing WT or mutant Atg30 proteins. White arrows indicate correct localization and yellow arrows indicate mislocalization, or in case of Atg8 localization, indicate absence of phagophore membrane elongation. Peroxisomes were labelled with BFP–SKL and vacuoles with FM4‐64. Scale bar, 5 μm. GFP, green fluorescent protein; WT, wild type.

Atg32, ScAtg32 and Atg36 use similar interaction mechanisms as Atg30. (A) Mitophagy experiments of WT (PPY12), Δatg5, Δatg32 (ϕ) and Δatg32 complemented with WT Atg32 (Atg32), Atg32 with a deletion of the sequence between the AIM and phosphosite required for Atg11 binding (Atg32WQVLSSS), Atg32 AIM mutant (Atg32W121A V124A), mutants of the Thr upstream of the Atg32 AIM (Atg32T119A) and mutants altered in the Ser required for Atg11 binding (Atg32S159A). The cells were grown in YPL medium …

… bind cargos and components of the autophagic machinery [ ]. In yeast, four receptors have been described: three in S. cereivisae , Atg19 (cytoplasm‐to‐vacuole targeting (Cvt) pathway), Atg32 (mitophagy) and Atg36 (pexophagy), and one in P. pastoris , Atg30 (pexophagy) [ , , , , ]. Atg19 interacts directly with the cargo (aminopeptidase I, Ape1) to form the Cvt complex, and subsequently with two autophagy proteins, Atg11 and Atg8 [ ]. Atg11 is a required protein for most selective autophagy …

Jean‐Claude Farré et al. EMBO Reports May 2013

Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19

Autophagy is the major pathway for the delivery of cytoplasmic material to the vacuole or lysosome. Selective autophagy is mediated by cargo receptors, which link the cargo to the scaffold protein Atg11 and to Atg8 family proteins on the forming autophagosomal membrane. We show that the essential kinase Hrr25 activates the cargo receptor Atg19 by phosphorylation, which is required to link cargo to the Atg11 scaffold, allowing selective autophagy to proceed. We also find that the Atg34 cargo receptor is regulated in a similar manner, suggesting a conserved mechanism.

Model representing the phospho‐regulation of the cargo receptor Atg19 and conceivably Atg34: Hrr25 and possibly Atg1 phosphorylate the cargo receptor in the Atg11 binding region. This promotes the interaction with Atg11.

Wild‐type, hrr25‐ts, and hrr25‐ts cells containing HRR25 on a centromeric plasmid were grown to mid‐log phase at 18°C followed by 2 and 4 h of log‐phase growth at 37°C. Ape1 processing was analyzed by Western blotting. atg19Δ and atg19Δ hrr25‐ts cells containing GFP‐Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37°C. GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg1‐D211A cells were analyzed for GFP‐Atg11 dot formation. Quantification of at least 70 cells …

Domain structure of Atg19. atg19Δ cells containing HTB‐Atg19 were grown to mid‐log phase. Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; lysine substitutions: gray; Atg11 binding region: green. atg19Δ cells containing GFP‐Atg19 wild‐type or mutants as indicated were grown to mid‐log phase. Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved …

… /SQSTM1 and NBR1 proteins , and the recently identified mitophagy receptor FUNDC1 . Saccharomyces cerevisiae Atg19 binds directly to its cargo Ape1 forming the so‐called Cvt complex. Subsequently, Atg19 mediates the transport of Ape1 to the vacuole by subsequent interactions with Atg11 and Atg8 . During bulk autophagy, Atg19 and the related protein Atg34 tether their respective cargo to the isolation membrane, thereby providing some selectivity to the otherwise non‐selective engulfment of cytoplasmic …

Thaddaeus Pfaffenwimmer et al. EMBO Reports August 2014

Pex3‐anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae

… pexophagy. We have isolated pex3 alleles blocked specifically in pexophagy that cannot recruit Atg36 to peroxisomes. Atg36 is recruited to mitochondria if Pex3 is redirected there, where it restores mitophagy in cells lacking the mitophagy receptor Atg32. Furthermore, Atg36 binds Atg8 and the adaptor Atg11 that links receptors for selective types of autophagy to the core autophagy machinery. Atg36 delivers peroxisomes to the preautophagosomal structure before being internalised into the vacuole with peroxisomes. We conclude that Pex3 recruits the pexophagy receptor Atg36. This reinforces the pivotal role played by Pex3 in coordinating the size of the peroxisome pool, and establishes its role in pexophagy in S. cerevisiae .

Atg11 and Atg8 bind Atg36. (A) Coimmunoprecipitation of Atg11 and Atg8 with Atg36. IP was performed in either C13–Atg36–PtA atg1Δ or C13–Mvp1–PtA atg1Δ cells containing plasmids expressing either HA–Atg11 or HA–Atg8 under control of the TPI1 promoter. Cells were grown for 24 h to post‐log phase in glucose medium followed by growth in oleate medium for a further 18 h. Cells were then grown under non‐starvation or starvation conditions for a further 2 h. IgG Sepharose beads were used …

… via Atg11. The Cvt receptors Atg19 and Atg34 also interact stably with Atg8 via an Atg8‐interacting motif (AIM), and this interaction is required for cargo delivery to the vacuole ( ; ). Likewise, in mammalian cells, known autophagy receptors bind to Atg8‐family orthologues via conserved LIRs (LC3‐interacting region), and these interactions are essential for function ( ; ; ). For one of these receptors, optineurin, a serine residue directly flanking the LIR can be phosphorylated …

Alison M Motley et al. The EMBO Journal June 2012

Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition

In addition to key roles in embryonic neurogenesis and myelinogenesis, γ‐aminobutyric acid ( GABA ) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6 K 1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm these processes in mammals, we examined the succinic semialdehyde dehydrogenase ( SSADH )‐deficient mouse model that accumulates supraphysiological GABA in the central nervous system and other tissues. Mutant mice displayed increased mitochondrial numbers in the brain and liver, expected with a defect in mitophagy, and morphologically abnormal mitochondria. Administration of rapamycin to these mice reduced m TOR activity, reduced the elevated mitochondrial numbers, and normalized aberrant antioxidant levels. These results confirm a novel role for GABA in cell signaling and highlight potential pathomechanisms and treatments in various human pathologies, including SSADH deficiency, as well as other diseases characterized by elevated levels of GABA .

… GABA only inhibits mitophagy and pexophagy in nutrient‐limited medium, but not ribophagy and general autophagy, is not yet clear. In proposing an explanatory model, we note that only Atg11‐dependent pathways are blocked by the addition of 10 mM GABA, as neither ribophagy nor general autophagy requires Atg11. In yeast, the phosphorylation‐dependent interaction of the mitophagy receptor, Atg32, and the pexophagy receptor, Atg36, with Atg11 is essential for the degradation of mitochondria …

Ronak Lakhani et al. EMBO molecular medicine April 2014

Mitochondria regulate autophagy by conserved signalling pathways

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae . During amino‐acid starvation, the autophagic response consists of two independent regulatory arms—autophagy gene induction and autophagic flux—and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1–Atg13 kinase complex to the pre‐autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors—mitochondrial dysfunction and autophagy inhibition—for ageing, cancerogenesis, and neurodegeneration.

… events. Towards this goal, we analysed the autophagic response in Δ atg1 , Δ atg7 , and Δ atg9 cells and Δ atg11 cells, which lack essential factors for general and selective autophagy, respectively ( ; ). Significantly, autophagic flux was completely blocked in Δ atg1 , Δ atg7 , or Δ atg9 cells ( ). We also observed a partial inhibition in autophagic flux in Δ atg11 cells during amino‐acid starvation, indicating that Atg11 has a more general role during amino‐acid starvation. However, ATG8 …

Martin Graef et al. The EMBO Journal June 2011

Nix is a selective autophagy receptor for mitochondrial clearance

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.

… cues and recruits Atg8 to stressed mitochondria. Interestingly, the LIR of Atg32 is also only partly required for mitophagy as Atg32 interacts with the Cvt pathway adaptor Atg11 independently of its LIR domain. This suggests that several signals might be essential for efficient mitophagy in yeast. Programmed mitochondrial clearance in reticulocytes is Nix‐dependent ( ; ) and Nix binding to LC3/GABARAP through LIR‐W35 might contribute to this process during reticulocyte maturation ( ). The effect …

Ivana Novak et al. EMBO Reports January 2010

ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy

Autophagy eliminates dysfunctional mitochondria in an intricate process known as mitophagy. ULK 1 is critical for the induction of autophagy, but its substrate(s) and mechanism of action in mitophagy remain unclear. Here, we show that ULK 1 is upregulated and translocates to fragmented mitochondria upon mitophagy induction by either hypoxia or mitochondrial uncouplers. At mitochondria, ULK 1 interacts with FUNDC 1, phosphorylating it at serine 17, which enhances FUNDC 1 binding to LC 3. A ULK 1‐binding‐deficient mutant of FUNDC 1 prevents ULK 1 translocation to mitochondria and inhibits mitophagy. Finally, kinase‐active ULK 1 and a phospho‐mimicking mutant of FUNDC 1 rescue mitophagy in ULK 1‐null cells. Thus, we conclude that FUNDC 1 regulates ULK 1 recruitment to damaged mitochondria, where FUNDC 1 phosphorylation by ULK 1 is crucial for mitophagy.

… of mitophagy receptors is modified by phosphorylation but the kinases responsible for these events are less clear . In yeast, Atg32 is phosphorylated at two sites, Ser‐114 and Ser‐119, and phosphorylation of Ser‐114 promotes Atg11–Atg32 interaction . Two mitogen‐activated protein kinases (MAPKs), namely Slt2 and Hog1, may be indirectly responsible for triggering the mitophagy signaling pathway . NIX is involved in autophagic degradation of mitochondria during reticulocyte maturation by an unknown …

Wenxian Wu et al. EMBO Reports May 2014

Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐ EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro . The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo . Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.

… . The Cvt complex interacts with Atg11 via binding of phosphorylated Atg19 and travels toward the pre‐autophagosomal structure where Atg19 binds lipidated Atg8 to associate the Cvt complex with the autophagy core machinery and the nascent Cvt vesicle . As the Cvt pathway utilizes a large proportion of the yeast autophagy protein machinery, it is considered a prototype system to study the structure and dynamics of selective autophagy in eukaryotes . Despite the fact that the Cvt vesicle carries …

Chiara Bertipaglia et al. EMBO Reports July 2016

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

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.

… is essential for autophagy, it has been proposed that Atg9 may have a pivotal role in supplying lipids to forming autophagosomes ( ; ). In yeast, Atg1, actin filaments, and a myosin‐like protein Atg11 have been shown to regulate the cycling of Atg9 ( ); however, the molecular mechanism underlying this process is not clear. In this study, we assessed whether myosin II might act as a motor protein in the regulation of Atg9 trafficking during autophagy. In mammalian cells, mAtg9 has been found to cycle from …

Hong‐Wen Tang et al. The EMBO Journal February 2011

Binding of the Atg1/ULK1 kinase to the ubiquitin‐like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo . Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)‐inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3‐interaction region (LIR)‐dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1–Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR‐dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.

… and V469 ( ). This interaction is direct as Atg1 and the binding domain of Atg13 purified from E. coli bind to each other in vitro in an F468‐ and V469‐dependent manner with almost 1:1 stoichiometry ( ). Co‐immunoprecipitation experiments confirmed that Atg13‐FV was unable to bind Atg1 ( ). Interestingly, its association with Atg11, Atg17 and Atg29 was also abolished, while the interaction with Vac8, a putative complex member, was only slightly reduced ( ; ). We concluded that F468 and V469 …

Claudine Kraft et al. The EMBO Journal September 2012
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