Regulation of Autophagy By Signaling Through the Atg1/ULK1 Complex

AbstractAutophagy is an intracellular degradation pathway highly conserved in eukaryotic species. It is characterized by selective or bulk trafficking of cytosolic structures—ranging from single proteins to cell organelles—to the vacuole or a lysosome, in which the autophagic cargo is degraded. Autophagy fulfils a large set of roles, including nutrient mobilization in starvation conditions, clearance of protein aggregates and host defence against intracellular pathogens. Not surprisingly, autophagy has been linked to several human diseases, among them neurodegenerative disorders and cancer. Autophagy is coordinated by the action of the Atg1/ULK1 kinase, which is the target of several important stress signaling cascades. In this review, we will discuss the available information on both upstream regulation and downstream effectors of Atg1/ULK1, with special focus on reported and proposed kinase substrates

Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores

The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and transmembrane Atg9 is completely unknown despite their importance in autophagy. In this study, we provide insights into the molecular role of these proteins by identifying and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates to autophagosomal membranes through lipid binding and independently from Atg9. Its interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn, disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy. Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and highlight the possible functional relevance of the phagophore-ER contact sites in phagophore expansion

Ykt6 mediates autophagosome-vacuole fusion

Studying the mechanism of autophagosome-vacuole fusion has proven difficult in live yeast cells. Developing a novel in vitro fusion assay, we identified Ykt6 as the missing R-SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) in this process and pinpoint the place of action of all four SNAREs involved. Parallel studies have confirmed our findings in other organisms

Assays to Monitor Autophagy in Saccharomyces cerevisiae

Autophagy is an intracellular process responsible for the degradation and recycling of cytoplasmic components. It selectively removes harmful cellular material and enables the cell to survive starvation by mobilizing nutrients via the bulk degradation of cytoplasmic components. While research over the last decades has led to the discovery of the key factors involved in autophagy, the pathway is not yet completely understood. The first studies of autophagy on a molecular level were conducted in the yeast Saccharomyces cerevisiae. Building up on these studies, many homologs have been found in higher eukaryotes. Yeast remains a highly relevant model organism for studying autophagy, with a wide range of established methods to elucidate the molecular details of the autophagy pathway. In this review, we provide an overview of methods to study both selective and bulk autophagy, including intermediate steps in the yeast Saccharomyces cerevisiae. We compare different assays, discuss their advantages and limitations and list potential applications

The Bacterial Translocon SecYEG Opens upon Ribosome Binding

In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist

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

Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores

The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and transmembrane Atg9 is completely unknown despite their importance in autophagy. In this study, we provide insights into the molecular role of these proteins by identifying and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates to autophagosomal membranes through lipid binding and independently from Atg9. Its interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn, disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy. Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and highlight the possible functional relevance of the phagophore-ER contact sites in phagophore expansion