Zvulun Elazar

Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.

Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles. This pathway is activated under environmental stress conditions, during development and in various pathological situations. In this study, we describe the role of reactive oxygen species (ROS) as signaling molecules in starvation‐induced autophagy. We show that starvation stimulates formation of ROS, specifically H2O2. These oxidative conditions are essential for autophagy, as treatment with antioxidative agents abolished the formation of autophagosomes and the consequent degradation of proteins. Furthermore, we identify the cysteine protease HsAtg4 as a direct target for oxidation by H2O2, and specify a cysteine residue located near the HsAtg4 catalytic site as a critical for this regulation. Expression of this regulatory mutant prevented the formation of autophagosomes in cells, thus providing a molecular mechanism for redox regulation of the autophagic process.

Lipophagy: Selective Catabolism Designed for Lipids

Until recently, degradation of lipid droplets (LDs) has been thought to take place in the cytosol by resident lipases. In a recent issue of Nature, Singh and coworkers describe the involvement of selective autophagy in the delivery of lipid droplets for lysosomal degradation.

A Role for NBR1 in Autophagosomal Degradation of Ubiquitinated Substrates

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.

Regulation of autophagy by ROS: physiology and pathology

Reactive oxygen species (ROS) are small and highly reactive molecules that can oxidize proteins, lipids and DNA. When tightly controlled, ROS serve as signaling molecules by modulating the activity of the oxidized targets. Accumulating data point to an essential role for ROS in the activation of autophagy. Be the outcome of autophagy survival or death and the initiation conditions starvation, pathogens or death receptors, ROS are invariably involved. The nature of this involvement, however, remains unclear. Moreover, although connections between ROS and autophagy are observed in diverse pathological conditions, the mode of activation of autophagy and its potential protective role remain incompletely understood. Notably, recent advances in the field of redox regulation of autophagy focus on the role of mitochondria as a source of ROS and on mitophagy as a means for clearance of ROS.

LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis

Autophagy, a critical process for bulk degradation of proteins and organelles, requires conjugation of Atg8 proteins to phosphatidylethanolamine on the autophagic membrane. At least eight different Atg8 orthologs belonging to two subfamilies (LC3 and GATE‐16/GABARAP) occur in mammalian cells, but their individual roles and modes of action are largely unknown. In this study, we dissect the activity of each subfamily and show that both are indispensable for the autophagic process in mammalian cells. We further show that both subfamilies act differently at early stages of autophagosome biogenesis. Accordingly, our results indicate that LC3s are involved in elongation of the phagophore membrane whereas the GABARAP/GATE‐16 subfamily is essential for a later stage in autophagosome maturation.

Biogenesis and cargo selectivity of autophagosomes.

Autophagy is a major catabolic pathway in eukaryotes, which is required for the lysosomal/vacuolar degradation of cytoplasmic proteins and organelles. Interest in the autophagy pathway has recently gained momentum largely owing to identification of multiple autophagy-related genes and recognition of its involvement in various physiological conditions. Here we review current knowledge of the molecular mechanisms regulating autophagy in mammals and yeast, specifically the biogenesis of autophagosomes and the selectivity of their cargo recruitment. We discuss the different steps of autophagy, from the signal transduction events that regulate it to the completion of this pathway by fusion with the lysosome/vacuole. We also review research on the origin of the autophagic membrane, the molecular mechanism of autophagosome formation, and the roles of two ubiquitin-like protein families and other structural elements that are essential for this process. Finally, we discuss the various modes of autophagy and highlight their functional relevance for selective degradation of specific cargos.

Does bafilomycin A1 block the fusion of autophagosomes with lysosomes

Bafilomycin A1 is a specific inhibitor of the vacuolar type H+-ATPase (V-ATPase) in cells, and inhibits the acidification of organelles containing this enzyme, such as lysosomes and endosomes. Recently, while editing and reviewing chapters on autophagy for Methods in Enzymology, we noticed repeated references to the effect of bafilomycin A1 in blocking the fusion of autophagosomes with lysosomes. Of course we have seen this in various research papers as well, but reading this routinely in chapters written by various people over a short period of time really caused this to stand out. Every one of these chapters referred to the paper by Yamamoto et al. In that paper, treatment with 100 nM bafilomycin A1 for 1 h blocks the fusion of autophagosomes with lysosomes in the rat hepatoma H-4-II-E cell line, based on data from electron microscopy. However, data from one of our labs noted an apparently different result in a relatively recent manuscript. Therefore, we decided to look into this more carefully.

Stepwise assembly of functionally active transport vesicles.

Budding of COP-coated vesicles (the likely carriers of newly synthesized proteins from the endoplasmic reticulum through the Golgi stack) from Golgi cisternae requires ADP-ribosylation factor (ARF), coatomer proteins from the cytosol, GTP, and fatty acyl-coenzyme A (CoA). The assembly of coated buds on the membranes requires coatomer, ARF, and GTP. When palmitoyl-CoA is added, membrane fission occurs at the coated bud, releasing coated vesicles. We show that COP-coated vesicles can be generated stepwise in vitro and isolated in a functionally active form, demonstrating that the minimal set of cytosolic components required for their formation as well as principal steps in their assembly have been identified.

Mechanisms of Autophagosome Biogenesis

Autophagy is a unique membrane trafficking process whereby newly formed membranes, termed phagophores, engulf parts of the cytoplasm leading to the production of double-membraned autophagosomes that get delivered to lysosomes for degradation. This catabolic pathway has been linked to numerous physiological and pathological conditions, such as development, programmed cell death, cancer, pathogen infection, neurodegenerative disorders, and myopathies. In this review, we will focus on recent studies in yeast and mammalian systems that have provided insights into two critical areas of autophagosome biogenesis - the source of the autophagosomal membranes, and the mechanisms regulating the fusion of the edges of the double-membraned phagophores to form autophagosomes.

Atg8: an autophagy-related ubiquitin-like protein family

Autophagy-related (Atg) proteins are eukaryotic factors participating in various stages of the autophagic process. Thus far 34 Atgs have been identified in yeast, including the key autophagic protein Atg8. The Atg8 gene family encodes ubiquitin-like proteins that share a similar structure consisting of two amino-terminal α helices and a ubiquitin-like core. Atg8 family members are expressed in various tissues, where they participate in multiple cellular processes, such as intracellular membrane trafficking and autophagy. Their role in autophagy has been intensively studied. Atg8 proteins undergo a unique ubiquitin-like conjugation to phosphatidylethanolamine on the autophagic membrane, a process essential for autophagosome formation. Whereas yeast has a single Atg8 gene, many other eukaryotes contain multiple Atg8 orthologs. Atg8 genes of multicellular animals can be divided, by sequence similarities, into three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3), γ-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which are present in sponges, cnidarians (such as sea anemones, corals and hydras) and bilateral animals. Although genes from all three subfamilies are found in vertebrates, some invertebrate lineages have lost the genes from one or two subfamilies. The amino terminus of Atg8 proteins varies between the subfamilies and has a regulatory role in their various functions. Here we discuss the evolution of Atg8 proteins and summarize the current view of their function in intracellular trafficking and autophagy from a structural perspective.