Autophagy Captures the Nobel Prize

Leading Edge

BenchMarks Autophagy Captures the Nobel Prize Sharon A. Tooze1 and Ivan Dikic2,3,* 1The

Francis Crick Institute, 1 Midland Road, NW1 1AT London, UK of Biochemistry II, School of Medicine, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany 3Buchmann Institute for Molecular Life Sciences, Goethe University, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2016.11.023 2Institute

This year’s Nobel Prize in Physiology or Medicine has been awarded to Yoshinori Ohsumi for the discovery of the molecular principles governing autophagy, an intracellular degradation pathway routed via lysosomes or vacuoles. It is a story of a simple yet insightful yeast genetic screen that revealed the inner circuitry of one of the most powerful quality-control pathways in cells. Autophagy, a lysosome-mediated intracellular degradation pathway, was discovered in the late 1950s. Cell biologists exploring the mammalian cell with then newly developed high-resolution transmission electron microscopic (TEM) techniques were the first to observe in cells lysosome-like membrane-bound structures that contained organelles like mitochondria. Later, those structures were called cytolysosomes and next autophagosomes. In 1963, the term autophagy was coined by Christian de Duve (for review see Eskelinen et al., 2011). At that time, it was impossible to imagine how broad and significant the influence of autophagy would turn out to be in the future. Autophagy is today recognized as a major quality-control pathway and metabolic regulation system in the cell. In essence, autophagy proceeds when a selected part of the cytosol is engulfed by a double membrane, forming the socalled autophagosome, which later fuses with lysosomes, digestive organelles of the cell, where a number of catabolic enzymes facilitate the breakdown of the cargo, enabling recycling of the generated metabolites. Autophagy can be driven by unspecific metabolic stimuli, such as starvation, or by highly specific signals facilitating the removal of damaged or superfluous proteins or organelles, thereby serving cellular homeostasis needs. The road to the discovery of the molecular basis of autophagy by Yoshinori Ohusumi is a story about importance of basic science, long-term dedication, and persistence that yielded unprecedented discoveries with broad significance to biology and medicine. The Royal Swedish Academy of Sciences now recognizes this

fascinating work by awarding the 2016 Nobel Prize in Medicine or Physiology to Yoshinori Ohsumi. A historical context is needed to appreciate the significance of Ohsumi’s remarkable work. From the 1950s to the 1980s, many groups had been observing the formation of vesicles that are able to deliver intracellular components to the lysosome (for review see Eskelinen et al., 2011). By the 1970s, biochemical analysis of this morphological phenomena in mammalian cells revealed the link between decreased amino acid levels and the formation of the autophagosome. The biochemical and morphological properties of autophagy continued to fascinate a small group of cell biologists through the 1980s during which time the suppressive potency of individual amino acids was reported, inhibitors were discovered, and the intracellular pathways and organelles involved were further explored (Ohsumi, 2014). At that time, there was no understanding of the molecular mechanisms underlying autophagy to begin to understand what proteins and lipids were required for autophagy in mammalian cells. Ohsumi approached this problem by studying autophagy in yeast, in which the lysosome-like vacuoles are end points of the autophagy pathway. In the 1980s, the first secretory pathway mutants were discovered by cell biologists working in the model system Saccharomyces cerevisiae, and progress was being made in understanding transport to and from the vacuole. Ohsumi identified autophagic bodies in nitrogen-deprived yeast S. cerevisiae and was the first to report that nutrient deficiency induced autophagic

degradation in yeast. A key point in his approach was the use of vacuolar protease inhibitors to allow for accumulation of intermediate autophagic bodies, the inner membrane-bound content of the double membrane autophagosome, inside the vacuole (Takeshige et al., 1992). Ohsumi’s lab characterized these structures by TEM and determined which proteases were required for their degradation (Figure 1). This seminal observation established the system and facilitated the first genetic screen for yeast autophagy mutants that was carried out in proteinase-deficient yeast cells to enable detection of the short-lived autophagic bodies. The mutants isolated were originally called ‘‘apg’’ (autophagy) (Tsukada and Ohsumi, 1993). Apg1-1 mutants were identified by their defect in accumulation of autophagic bodies, and the Ohsumi lab observed that they lost viability much faster under conditions of nutrient deprivation. A further viability screen then elegantly identified 75 mutants that fell into another 15 complementation groups. With this groundbreaking work, the Ohsumi lab identified the first autophagy genes and showed that there are at least 15 needed in yeast. Others were soon to follow, as similar approaches were ongoing in Thumm’s lab in Germany, who identified three new genes called AUT (Thumm et al., 1994), and in Klionsky’s lab on the cytoplasm-to-vacuole (CVT) pathway (Harding et al., 1995). The commonalities between the nutrient-sensitive autophagy and constitutive CVT pathways were discovered through analysis of the apg complementation groups (Scott et al., 1996). By 2003, the number of autophagy genes required for survival

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Figure 1. Autophagosomes Transmission electron microscopy was fundamental for the identification of the cytosolic autophagosomes in yeast by Yoshinori Ohsumi’s group in 1994 (reproduced with permission from Figure 2 in Baba et al., J. Cell Bio. 124, 903–913).

in yeast grew to 27 and the nomenclature for the genes designated APG, AUT, CVT, GSA, PAG, PAZ, and PDD was unified to ATG (Klionsky et al., 2003). Today, the number of autophagy-related genes (ATGs) has grown to around 40 in yeast with 15 constituting the core machinery of autophagosome formation and the others being required for modulating the core machinery or selective modes of autophagy. The identification of the yeast ATG genes triggered an almost explosive increase in knowledge not only regarding the mechanistic details of the autophagosomal cargo uptake and degradation in the vacuole/lysosome but also concerning the regulation of the entire process, its physiological meaning, and involvement in various diseases. At a molecular level, progress on understanding the regulation of autophagy began with the characterization of Apg1 (Atg1), as a novel type of Ser/Thr kinase, being a homolog of the C. elegans unc-51 protein, followed by understanding of its control by TOR (target of rapamycin) consistent with mTOR’s role in mammalian cells (Ohsumi, 2014). In yeast, the Atg1 serine-threonine kinase complex (including Atg1, Atg13, Atg17, Atg29, and Atg31) is thought to be the most upstream regulator of autophagy. The second conserved kinase 1434 Cell 167, December 1, 2016

complex in this context is a lipid kinase complex containing Vps34, also known as the class III phosphoinositide 3-kinase (PtdIns 3)-kinase complex I in mammals, which consists of a catalytic subunit, Vps34, the regulatory subunit p150 (homolog of yeast Vps15), Beclin 1 (yeast Vps30), and Atg14. Soon after the initial discovery of the autophagy genes, two catalytic conjugation cascades that are linked to each other and essential for autophagosomal formation were identified by Ohsumi, Mizushima, and colleagues (Mizushima et al., 1998; Ichimura et al., 2000). In principle, both systems are analogous to the ubiquitination conjugation system, which was already well described at the time. Atg12 and Atg8 function as ubiquitin-like modifiers in this context and are activated by the common E1-like protein Apg7 (Atg7) and transferred to the E2-like enzymes Atg10 and Atg3, respectively. So far, Atg5 (Apg5) is the only specific target known for Atg12. In contrast, Atg8 is covalently attached to a membrane phospholipid, phosphatidylethanolamine (PE), rather than other proteins. Meanwhile, the entire lipidation cascade was revealed, showing that Atg8 first needs to be processed by a cysteine protease, Atg4, followed by the action of Atg7/ Atg3 enzymes and the action of the Atg5-Atg12 conjugate functioning as an E3-like enzyme in the lipidation reaction. Lipidated Atg8 marks the pre-autophagosomal structure, located near the vacuole, thus generating a critical signal and a marker of the biogenesis of these vesicles. Both conjugation cascades are conserved and critical in mammals. Today, it is well established that Atg8-mediated lipidation is central to autophagosome formation. As shown in a landmark publication by Yoshimori and colleagues in 2000, Atg8 homologs of the LC3/ GABARAP protein family take over this critical role in mammalian cells (Kabeya et al., 2000). They showed that lipidated LC3/GABARAP proteins localize to the autophagosomal membrane, which provided the first good marker for autophagosomal membranes in mammals, paving the way for hundreds of studies in the past two decades. The original publication by Yoshimori’s group is until today the most cited manuscript in the autophagy com-

munity, supporting the widespread role of LC3 in multiple autophagy pathways in higher eukaryotes. In addition, Mizushima and Ohsumi generated a transgenic mouse systemically expressing GFPLC3, enabling in vivo fluorescence microscopy and visualization of the autophagosome, which was freely shared within the scientific community and has fueled numerous further studies (Mizushima et al., 2004). Besides the ATG12-ATG5 and LC3-PE conjugation systems, other Atg proteins have been shown to be critical in mammals including the ULK complex, the PtdIns 3-Kinase complex, and WIPI2 (reviewed in Ohsumi, 2014). This preservation of the major functional autophagy units across species enabled a fast transfer of mechanistic understanding of autophagy from yeast to multicellular organisms. In particular, early on it was shown by Levine’s group that Beclin 1 can functionally substitute for Atg6 and thereby rescue nitrogenstarved yeast (Liang et al., 1999). Furthermore, they showed that bec-1, the Beclin 1 homolog in C. elegans, was shown along with other Atg proteins including Atg1, 7, 8, and 18, to be required for autophagy during dauer development and lifespan extension (Ohsumi, 2014). Moving from model systems to mice, Mizushima’s group generated the knockout mouse for Atg5 and observed that those mice died within 24 hr of birth, demonstrating that autophagy is important for survival during the neonatal stage of development in mice. It is one of the remarkable signs of the strength of the autophagy community that both the GFP-LC3 transgenic and several knockout mice of ATG genes have been shared with many labs around the world and have paved the road to new discoveries regarding the physiological significance of autophagy in mammals. Up to now, more than a dozen Atg genes have been deleted in mice (for review, see Ohsumi, 2014) and the phenotype of these mice demonstrates the multilayered role of autophagy genes in physiological and pathophysiological conditions in individual cells and tissues. In the past two decades, autophagy has been connected to multiple human diseases including cancer, neurodegenerative diseases, aging, metabolic disorders, inflammation, infectious diseases, and others and this accumulated

sharing and co-operation that was nurtured within this community. Finally, Yoshinori Ohsumi’s work gives a remarkable example to funding agencies how original research using the yeast system can have profound consequences reaching out to cell biology and (patho) physiology. REFERENCES Eskelinen, E.-L., Reggiori, F., Baba, M., Kova´cs, A.L., and Seglen, P.O. (2011). Autophagy 7, 935–956. Harding, T.M., Morano, K.A., Scott, S.V., and Klionsky, D.J. (1995). J. Cell Biol. 131, 591–602. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., et al. (2000). Nature 408, 488–492.

Figure 2. The Impact of Autophagy on Health A simplified representation of autophagy delivery of cargoes to the lysosome. Autophagosomal membranes are able to surround specific cargoes including damaged mitochondria, protein aggregates, pathogens, or defective organelles and deliver them to the lysosome to regulate cell homeostasis and defend cellular integrity. Deregulation of autophagy results in the development of numerous human diseases.

knowledge is channeled toward targeting autophagy in disease treatments (Rubinsztein et al., 2012). This compelling amount of new information related to the role of autophagy in human pathologies follows up on an early discovery by Levine and colleagues demonstrating a link between Beclin 1 with the induction of autophagy in human cells and the association with human malignancies (Liang et al., 1999). In this context, overwhelming evidence emerged conceptualizing autophagy not only as an unspecific catabolic process providing energy and nutrients under starvation conditions, but also as a guardian of cellular homeostasis and integrity. A central function of autophagy in the mammalian system is cleaning the cell interior from unwanted and potentially harmful cellular components that disturb cellular homeostasis. Selective autophagy pathways that target specific nonfunctional cellular components, such as

misfolded protein aggregates or dysfunctional mitochondria, but also intruders such as intracellular bacteria, have moved into the focus of autophagy researchers. Critical insight into the selectivity of the autophagy pathway has been provided by studies deciphering the network of LC3/GABARAP-binding proteins that function as autophagy receptors and adaptors, facilitating the specific recognition and targeting of cargo to autophagosomes (Stolz et al., 2014). In the still-growing field of autophagy, which has seen an enormous boost after the pioneering work of Ohsumi and colleagues, many groups across the world have significantly contributed to further our understanding of the pivotal role of autophagy in metabolic homeostasis, in regulation of vital processes in the cell, and in guarding cellular integrity (Figure 2). We would like to give credit to all of them and particularly acknowledge the spirit of

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