Tamotsu Yoshimori

LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing

Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule‐associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3‐I and ‐II, were produced post‐translationally in various cells. LC3‐I is cytosolic, whereas LC3‐II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3‐II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3‐II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3‐I is formed by the removal of the C‐terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3‐I into LC3‐II. The amount of LC3‐II is correlated with the extent of autophagosome formation. LC3‐II is the first mammalian protein identified that specifically associates with autophagosome membranes.

Methods in Mammalian Autophagy Research

Autophagy has been implicated in many physiological and pathological processes. Accordingly, there is a growing scientific need to accurately identify, quantify, and manipulate the process of autophagy. However, as autophagy involves dynamic and complicated processes, it is often analyzed incorrectly. In this Primer, we discuss methods to monitor autophagy and to modulate autophagic activity, with a primary focus on mammalian macroautophagy.

The role of autophagy during the early neonatal starvation period

At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. Here, we show that neonates adapt to this adverse circumstance by inducing autophagy. Autophagy is the primary means for the degradation of cytoplasmic constituents within lysosomes. The level of autophagy in mice remains low during embryogenesis; however, autophagy is immediately upregulated in various tissues after birth and is maintained at high levels for 3–12 h before returning to basal levels within 1–2 days. Mice deficient for Atg5, which is essential for autophagosome formation, appear almost normal at birth but die within 1 day of delivery. The survival time of starved Atg5-deficient neonates (∼ 12 h) is much shorter than that of wild-type mice (∼ 21 h) but can be prolonged by forced milk feeding. Atg5-deficient neonates exhibit reduced amino acid concentrations in plasma and tissues, and display signs of energy depletion. These results suggest that the production of amino acids by autophagic degradation of ‘self’ proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.

How to interpret LC3 immunoblotting.

Microtubule-associated protein light chain 3 (LC3) is now widely used to monitor autophagy. One approach is to detect LC3 conversion (LC3-I to LC3-II) by immunoblot analysis because the amount of LC3-II is clearly correlated with the number of autophagosomes. However, LC3-II itself is degraded by autophagy, making interpretation of the results of LC3 immunoblotting problematic. Furthermore, the amount of LC3 at a certain time point does not indicate autophagic flux, and therefore, it is important to measure the amount of LC3-II delivered to lysosomes by comparing LC3-II levels in the presence and absence of lysosomal protease inhibitors. Another problem with this method is that LC3-II tends to be much more sensitive to be detected by immunoblotting than LC3-I. Accordingly, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3-II for ratio determinations, may not be appropriate, and rather, the amount of LC3-II can be compared between samples.

Inhibition of Macroautophagy Triggers Apoptosis

ABSTRACT Mammalian cells were observed to die under conditions in which nutrients were depleted and, simultaneously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/Beclin 1-1, Atg10, or Atg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition. Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein, ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mitochondrial transmembrane potential were beyond the point of no return and inexorably died even under optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell death pathways.

The role of Atg proteins in autophagosome formation.

Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production.

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohns disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1β and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1β. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1β and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.

A protein conjugation system essential for autophagy

Autophagy is a process for the bulk degradation of proteins, in which cytoplasmic components of the cell are enclosed by double-membrane structures known as autophagosomes for delivery to lysosomes or vacuoles for degradation. This process is crucial for survival during starvation and cell differentiation. No molecules have been identified that are involved in autophagy in higher eukaryotes. We have isolated 14 autophagy-defective (apg) mutants of the yeast Saccharomyces cerevisiae and examined the autophagic process at the molecular level. We show here that a unique covalent-modification system is essential for autophagy to occur. The carboxy-terminal glycine residue of Apg12, a 186-amino-acid protein, is conjugated to a lysine at residue 149 of Apg5, a 294-amino-acid protein. Of the apg mutants, we found that apg7 and apg10 were unable to form an Apg5/Apg12 conjugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-E1-like enzyme. This conjugation can be reconstituted in vitro and depends on ATP. To our knowledge, this is the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system. Furthermore, Apg5 and Apg12 have mammalian homologues, suggesting that this new modification system is conserved from yeast to mammalian cells.

Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein, Tandem Fluorescent-Tagged LC3

During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric Red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.

LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation.

Rat LC3, a homologue of yeast Atg8 (Aut7/Apg8), localizes to autophagosomal membranes after post-translational modifications. The C-terminal fragment of LC3 is cleaved immediately following synthesis to yield a cytosolic form called LC3-I. A subpopulation of LC3-I is further converted to an autophagosome-associating form, LC3-II. Because yeast Atg8 is conjugated with phosphatidylethanolamine (PE) by a ubiquitin-like system, it has been hypothesized that LC3 is modified in a similar manner. Here, we show that [14C]-ethanolamine was preferentially incorporated into LC3-II, suggesting that LC3-II is a PE-conjugated form. LC3-II can be a substrate of mammalian Atg4B, a homologue of yeast Atg8-PE deconjugase, supporting the idea that LC3-II is LC3-PE. Moreover, two other mammalian homologues of yeast Atg8, γ-aminobutyric-acid-type-A-receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE16) also generate form II, which are recovered in membrane fractions. Generation of the form II correlates with autophagosome association of GABARAP and GATE16. These results suggest that all mammalian Atg8 homologues receive a common modification to associate with autophagosomal membrane as the form II.