The initial steps of autophagy - Atg1 and its targets

Autophagie ist ein eukaryotischer Abbaumechanismus für langlebige Proteine und Organellen, der unter nährstoffarmen Bedingungen hochreguliert wird. Vorangehende Studien bestätigten bereits die Schlüsselfunktion der Atg1 Kinase in der Aktivierung von Autophagie. Bisher wurden allerdings noch keine Substrate von Atg1 beschrieben. Vor kurzem wurde eine Konsensussequenz für durch Atg1 modifizierte Peptide mittels in vitro Methoden definiert. Dies ermöglichte die Identifizierung von Atg2, Atg9 und Atg18 als wahrscheinliche Substrate der Atg1 Kinase. In dieser Arbeit präsentieren wir einen Ansatz um diese Hypothese mit in vivo Methoden zu bestätigen. Dafür verwendeten wir quantitative Massenspektrometrie und einen neuen Protein-Protein Interaktionsassay, der auf sogenanntem „enzymatischem Tagging“ basiert. Unsere Experimente zeigen, dass Atg9 und Atg2 Substrate von Atg1 sind. Zusätzlich konnten wir eine auf unseren in vivo Daten basiernde Atg1 Konsensussequenz definieren.Autophagy is a degradation pathway of organelles and long lived proteins which becomes activated upon starvation conditions in all eukaryotes. Previous studies have validated the key role of the protein kinase Atg1 in the activation of autophagy; however, so far no targets have been described. Recently, a consensus motif for sites that are modified by Atg1 has been determined by in vitro kinase assays and bioinformatic analyses. Furthermore, the autophagy related factors Atg2, Atg9 and Atg18 have been proposed as putative Atg1 substrates. Here we present an approach to validate these observations in vivo. To do so, we used mass spectrometry and a novel protein-protein proximity assay and that based on stable enzymatic modifications. Our analysis showed that Atg9 and Atg2 are indeed substrates of Atg1. Moreover, we defined an Atg1 consensus motif based on in vivo data

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. The EMBO Journal (2012) 31, 3691-3703. doi:10.1038/emboj.2012.225; Published online 10 August 201

Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase

Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes.ISSN:1097-2765ISSN:1097-416

Proceedings Of The 23Rd Paediatric Rheumatology European Society Congress: Part Two

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