Yoshinori Ohsumi

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.

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.

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice

Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene

Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus-induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.

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.

A ubiquitin-like system mediates protein lipidation

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.

Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae.

Autophagy in the yeast is similar to that in mammalian cells. A mutant designated as apg1 (autophagy) defective in accumulation of autophagic bodies in the vacuoles was isolated by selection using a light microscope from a mutagenized proteinase‐deficient strain. In the apg1 strain, which has normal vacuolar proteinases, nitrogen starvation did not induce protein degradation. The apg1 mutant lost its viability faster than wild‐type cells during nitrogen starvation. By using the loss of viability as a first screening test, 75 other apg mutants were selected. These apg mutants including apg1 fell into 15 complementation groups. Genetic analyses of representative apg mutants revealed that they all had single recessive chromosomal mutations. Strains with each apg mutation were defective in protein degradation in the vacuoles induced by nitrogen starvation and homozygous diploids for each apg mutation did not sporulate. These results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved in autophagy in yeast.

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.

Dynamics and diversity in autophagy mechanisms: lessons from yeast

Autophagy is a fundamental function of eukaryotic cells and is well conserved from yeast to humans. The most remarkable feature of autophagy is the synthesis of double membrane-bound compartments that sequester materials to be degraded in lytic compartments, a process that seems to be mechanistically distinct from conventional membrane traffic. The discovery of autophagy in yeast and the genetic tractability of this organism have allowed us to identify genes that are responsible for this process, which has led to the explosive growth of this research field seen today. Analyses of autophagy-related (Atg) proteins have unveiled dynamic and diverse aspects of mechanisms that underlie membrane formation during autophagy.

A unified nomenclature for yeast autophagy-related genes

The authors would like to thank Drs. Jan A.K.W. Kiel, Ida J. van der Klei, Beth Levine, Fulvio Reggiori, and Takahiro Shintani for helpful comments on the manuscript, and the many researchers in the yeast field who have agreed to changes in the standard names of various genes.