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Autophagywins the2016NobelPrize inPhysiologyor Medicine: Breakthroughs in baker’s yeast fueladvances in biomedical researchBeth Levinea,b,c,1 and Daniel J. Klionskyd

Edited by Inder Verma, The Salk Institute for Biological Studies, La Jolla, CA, and approved December 7, 2016 (received for reviewDecember 2, 2016)

Autophagy is an ancient pathway in which parts of eukaryotic cells are self-digested within the lysosome orvacuole. This process has been studied for the past seven decades; however, we are only beginning to gaina molecular understanding of the key steps required for autophagy. Originally characterized as a hormonaland starvation response, we now know that autophagy has a much broader role in biology, includingorganellar remodeling, protein and organelle quality control, prevention of genotoxic stress, tumorsuppression, pathogen elimination, regulation of immunity and inflammation, maternal DNA inheritance,metabolism, and cellular survival. Although autophagy is usually a degradative pathway, it also participatesin biosynthetic and secretory processes. Given that autophagy has a fundamental role in many essentialcellular functions, it is not surprising that autophagic dysfunction is associated with a wide range of humandiseases. Genetic studies in various fungi, particularly Saccharomyces cerevisiae, provided the key initialbreakthrough that led to an explosion of research on the basic mechanisms and the physiological connec-tions of autophagy to health and disease. The Nobel Committee has recognized this breakthrough by theawarding of the 2016 Nobel Prize in Physiology or Medicine for research in autophagy.

On October 3, 2016, the Nobel Prize in Physiology orMedicinewas awarded to Yoshinori Ohsumi for “discov-eries of the mechanisms for autophagy.” Just a fewweeksearlier, at anacceptancespeech for the2016PaulJanssen Award, Yoshinori Ohsumi stated that althoughhe performs research in a simple organism—baker’syeast—he always hoped his research would have an im-pact upon human health. Indeed, Ohsumi’s discoveries,along with those of others working simultaneously onautophagy-related pathways in yeast, have provided apowerful molecular tool kit that thousands of scientistsare now using worldwide to decipher the roles of au-tophagy in mammalian health and disease. The story ofthis year’s Nobel Prize represents a better-than-textbookexampleofwhyunbridleddiscovery in fundamental basicscience is crucial for opening new frontiers in medicine.

Autophagy Probably Evolved Billions of Years AgoDerived from the Greek and meaning to eat (“phagy)oneself (“auto”), the term autophagy refers to a lyso-somal degradation pathway of self-digestion (Fig. 1) (1).

This process involves dynamic membrane rearrange-ments to sequester cargo for delivery to the lysosome,where the sequestered material is degraded andrecycled. We now know that autophagy can benonselective (such as during nutrient deprivation) orexquisitely selective (such as when damaged organ-elles or intracellular pathogens are degraded). Themolecular machinery of autophagy (first discovered inyeast as described below) is present in the genome ofevery eukaryotic organism that has been sequencedto date, but not in that of any prokaryotes. Thus, theorigins of autophagy are inextricably linked to co-evolutionary events essential for the emergence ofeukaryotic life.

Autophagy Was First Described in MammalianCells in the 1950sThe history of autophagy research began when sci-entists looked at mammalian cells under the electronmicroscope. According to Christian de Duve (Nobel

aCenter for Autophagy Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390;bDepartment of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390; cHoward Hughes Medical Institute, Universityof Texas Southwestern Medical Center, Dallas, TX 75390; and dLife Sciences Institute, University of Michigan, Ann Arbor, MI 48109This article is part of a series of articles in PNAS highlighting the discoveries and profiling the award winners of the Nobel Prize. Articles about the2016 Nobel Laureates in Physics and in Chemistry can be found at 10.1073/pnas.1620134114 and 10.1073/pnas.1619330114.Author contributions: B.L. and D.J.K. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: beth.levine@utsouthwestern.edu.

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Laureate, 1974, discoverer of the lysosome) (2), the word “au-tophagy” was invented at the Ciba Foundation Symposium onLysosomes in London in 1963. At the meeting, Alex Novikoffdescribed his studies characterizing “cytolysosomes,” acid phos-phatase-positive structures that contained degrading mitochon-dria, endoplasmic reticulum membranes, ribosomes, and othercytoplasmic contents, and de Duve then proposed the termautophagy to denote this function of lysosomes in self-eating (thisprocess is now termed macroautophagy, but we use autophagyfor simplicity). In 1957, autophagosome-like structures (i.e., dou-ble-membrane vesicles) were found to be enriched in the de-veloping newborn rat kidney (3), and later, large-scale autophagywas described during insect metamorphosis (4). In 1962, Ashfordand Porter (5) and Novikoff and Essner (6) showed that autopha-gosomes were increased in rat livers exposed to glucagon andstarvation, respectively. In 1977, Pfeifer (7) and Mortimore andSchworer (8) demonstrated that insulin and amino acids, re-spectively, suppressed hepatic autophagy. In 1982, Seglen andGordon (9) performed the first biochemical analysis of autophagyand identified the pharmacological reagent 3-methyladenine asan autophagy inhibitor. Subsequently, the groups of Seglen (10),Meijer (11), and Codogno (12) first noted that protein kinases,phosphatases, and heterotrimeric G proteins can regulate au-tophagy. In 1988, Seglen’s team identified the phagophore (theinitial sequestering vesicle that develops into the autophago-some) (13). In 1992, Meijer’s group (14) showed that peroxisomespreferentially undergo autophagic degradation in rat hepatocyteswhen deprived of their substrates [consistent with earlier studieson peroxisomal degradation in methylotrophic yeasts (15)].

These key early findings established the existence of au-tophagy in mammalian cells; its regulation by nutrient status,nutrient-sensing hormones, kinases, and phosphatases; its po-tential for selective cargo degradation; and its increase in certainstress and developmental conditions. Remarkably, a review articleby de Duve and Wattiaux in 1966 (16) presaged much of whatwe know today about the functions of autophagy. These in-cluded a role in “nutrition under unfavorable conditions of foodsupply through piecemeal self digestion,” “cellular differentia-tion and metamorphosis,” “intracellular scavenging as part ofthe self-rejuventation of long-lived cells,” and the “self-clearanceof dead cells.” However, there was one major problem—howcould this (or other) functionality of autophagy be proved by

morphological observations? Herein lies the basis for the impor-tance of describing the conserved genetic machinery of au-tophagy in yeast.

Enter Ohsumi and Other Yeast Researchers: The Dawn ofthe Genetic Era of Autophagy ResearchIn the early 1990s, several laboratories began studying non-selective and selective autophagy in yeasts. In 1992, Ohsumi andcolleagues (17) described the presence of “autophagic bodies” inthe vacuole (the analog of the lysosome) of protease-deficientSaccharomyces cerevisiae devoid of nutrients—the first morpho-logical characterization of nonselective autophagy in yeast. In thesame issue of the Journal of Cell Biology, Klionsky et al. (18) de-scribed the import of aminopeptidase I into the vacuole of Sac-charomyces cerevisaie—the first characterization of selectiveautophagy in yeast. Shortly thereafter, Dunn and colleagues (19)described a form of selective autophagy in the methylotrophicyeasts, Pichia pastoris and Hansenula polymorpha, involving theselective degradation of peroxisomes in the vacuole.

These morphological and biochemical characterizations setthe stage for nearly parallel efforts in three laboratories to isolatemutants in S. cerevisiae and clone the genes encoding the yeastautophagy machinery, now referred to as the autophagy-related(ATG) genes (20); similar efforts were undertaken in several labo-ratories working with the methylotrophic yeasts (21). Ohsumi’sgroup (22) performed a genetic screen to isolate mutants thataccumulate autophagic bodies in the vacuole and show de-creased viability during nitrogen starvation (originally known asthe apgmutants); Thumm et al. (23) performed a genetic screen toisolate yeasts deficient in protein uptake for degradation in thevacuole (originally known as the aut mutants); and Klionsky’sgroup (24) performed a screen for mutants deficient in the deliveryof a resident vacuolar hydrolase from the cytoplasm to the vacuole(originally known as the cvt mutants).

In October 1996, the first yeast autophagy gene, APG5 (nowknown as ATG5), was reported by Ohsumi and colleagues (25).Shortly after, in February 1997, Thumm’s group (26) reportedthe cloning of AUT1 (now known as ATG3), which encodes anE2-conjugating enzyme involved in the modification of theubiquitin-like protein Atg8. In 1998, Mizushima et al. (27) describeda protein conjugation system in yeast, the Atg12–Atg5 system thatrequires a ubiquitin activating E1-like enzyme, Atg7, and in 1999,three additional groups (28–30) reported in back-to-back papersthe cloning and characterization of yeast ATG7. In 2000, Ichimuraet al. (31) reported that the Atg7 E1-like enzyme conjugates Atg8 tophosphatidylethanolamine, establishing a role for protein lipidationin membrane dynamics during autophagy.

Taken together, between 1996 and 2000, the various labora-tories working with yeasts cloned genes encoding componentsthat function at different stages of autophagy including initiation(involving the Atg1 serine/threonine kinase complex and theVps34 lipid kinase complex) as well as phagophore expansion andautophagosome maturation (involving the two ubiquitin-likeprotein conjugation systems). From this period until the present,extensive studies (particularly in S. cerevisiae) using moleculargenetic, biochemical, and structural approaches have continuedto teach us about the workings of the core autophagy machineryand have identified additional components critical for varioustypes of selective autophagy.

Fig. 1. Ultrastructure of autophagy in yeast and mammalian cells.(Left) Electron micrograph of a yeast (S. cerevisiae) cell withautophagic bodies in the vacuole during nitrogen starvation.Reproduced with permission from ref. 54. (Right) Electron micrographof a forming autophagosome in an amino acid-starved beclin1-deficient human breast cancer cell expressing exogenous Beclin1. Courtesy of X. H. Liang, N. Furuya, Y. Wei, and B. Levine. (Scalebars, 1 μm.)

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The Dawn of the Genetic Era of Mammalian AutophagyResearchStudies in yeast were crucial to elucidate the molecular mecha-nisms of autophagosome formation. Early yeast genetic screensalso established that autophagy is essential for survival duringstarvation and for the yeast differentiation process of sporulation.However, as yeasts are unicellular organisms, the discovery of themolecular machinery of autophagy in more complex eukaryotes(especially mammals) was critical to define the functions of au-tophagy in health and disease.

Throughout the 1990s, several vertebrate genes were clonedthat shared a high degree of sequence homology with essentialyeast autophagy genes, including bovine PIK3C3/VPS34 (32), ratMAP1LC3/LC3 (33), mouse and human Ulk1/ULK1 (34, 35), humanATG5 (36, 37), human ATG12 (37), and human VPS30/ATG6(beclin 1) (38). In 1999, Levine’s group (39) provided the firstdemonstration of an autophagic function of a mammalian protein;Beclin 1, the human ortholog of yeast Vps30/Atg6, rescuedautophagy in VPS30/ATG6-deficient yeasts and in autophagy-deficient breast cancer cells lacking one copy of beclin 1. Over thenext several years, it was demonstrated that virtually all of theyeast autophagy genes are functionally conserved throughoutmetazoan evolution.

The evolutionary conservation of autophagy genes providedan unparalleled opportunity to perform reverse genetics in a widevariety of model organisms—ranging from flies to plants to wormsto mice—to probe the function of autophagy in diverse biologicalprocesses. In 2003, Heintz’s group (40) and Levine’s group (41)independently reported the first autophagy gene knockoutmouse; homozygous deletion of beclin 1 is embryonically lethaland heterozygous deletion of beclin 1 results in increased spon-taneous tumorigenesis [consistent with the earlier reported tumorsuppressor activity of beclin 1 in breast cancer cells and its com-mon allelic loss in human ovarian and breast cancer (39, 42)]. Thesame year, Levine’s group showed that autophagy genes wereessential for life span extension and dauer development in nem-atodes (43). In 2004, Mizushima’s group (44) reported theknockout of the first autophagy ubiquitin-like conjugation systemgene, Atg5; these mice (and additional targeted mutant micelacking other autophagy conjugation system genes) survived tobirth but died shortly thereafter, presumably due to an inability toadapt to a postnatal starvation period. Also in 2004, three groupsreported a role for the autophagy machinery in degrading med-ically important bacteria that invade intracellularly, includingMycobacterium tuberculosis (45), group A Streptococcus (46), andShigella flexneri (47).

Fig. 2. Attendees of the first Gordon Research Conference on “Autophagy in Stress, Development, and Disease” (Colby College, Maine, June22–27, 2003). In the front row (positions 10–12) are Daniel J. Klionsky, the vice chair; Beth Levine, the chair; and Yoshinori Ohsumi, the 2016Nobel Laureate in Physiology or Medicine.

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These early studies were soon followed by a plethora of ad-ditional genetic analyses in various metazoan organisms, whichunequivocally established a crucial role for the autophagy ma-chinery in differentiation and development, nutrient and energyhomeostasis, stress adaptation, tumor suppression, innate and adap-tive immunity, metabolism, regulation of inflammation, and protectionagainst cardiac and skeletal diseases, neurodegeneration, and ag-ing (48–50). These roles stem from the ability of autophagy to de-grade unwanted or harmful substrates (largely via selectiveautophagy), to recycle cytoplasmic constituents during environmen-tal stress (largely through nonselective autophagy), and from otherhomeostatic functions of the autophagy machinery. Importantly, it isnow clear that mutations in genes encoding core components thatare required for autophagosome formation are associated withhuman disease (51, 52).

Yeast and Mammalian Autophagy Researchers UniteThe first Gordon Research Conference on autophagy, held in2003, brought together researchers from North and SouthAmerica, Europe, the Middle East, and Asia (Fig. 2). Its partici-pants included nearly all of the living autophagy researchersmentioned above, as well as many other senior leaders and risingstars in the field. Yoshinori Ohsumi gave the keynote lecture andthrew the first pitch at a “yeast versus mammals” softball gameheld during an afternoon break. The transnational and trans-kingdom nature of the meeting united participants with diverseexpertise but who shared a common passion for autophagy, thuslaying the groundwork for enduring scientific collaborations thatmarkedly accelerated the pace of discovery in the field. As one ofmany examples, Yoshimori’s group (53) had discovered that GFP-LC3 was a useful marker of autophagosomes; he generouslyshared his reagents, and fluorescent-tagged versions of Atg8/LC3orthologs have been used worldwide ever since to monitorautophagosome numbers in a wide range of species.

The High Noon of Autophagy ResearchThanks to the elucidation of the genetic machinery and molecularmechanisms for autophagy in yeast (and all of the subsequentwork in more complex eukaryotes that has built upon this foun-dation), we have entered an exciting new era in autophagy re-search. We have made major inroads into understanding howautophagy is regulated, what the core autophagy machinery lookslike structurally and how it works, what autophagy is doing to keepus healthy (or in more rare circumstances, make us ill), and how wecan manipulate autophagy pharmacologically or via lifestyle in-terventions to prevent and/or treat aging and many commondiseases. We are also learning that the genetic machinery ofautophagy participates in other trafficking and signaling pathwaysto control mammalian physiology, thus further expanding thelong-term biomedical impact of the original yeast genetic screens.Although not directly linked to the yeast screens for genes re-quired for autophagosome formation, the recent revolution inidentifying genes required for selective autophagy (many of whichare mutated in human diseases, particularly cancer and neuro-degenerative disorders) also holds great promise for strategies toaugment autophagy to protect against disease.

To be sure, there are many questions yet to be answered aboutthe basic mechanisms of autophagy (and other autophagy gene-dependent processes), its roles in disease, and its potential as apathway for drug targeting. Nonetheless, it is quite possible thatin our lifetime, or at least that of our children, we will witness therealization of Yoshinori Ohsumi’s hope—the eloquent moleculardissection of a previously underappreciated cell biology processin yeast will likely ultimately fuel groundbreaking advances inhuman health.

AcknowledgmentsThe authors are supported by NIH Grants U19 AI199725 and RO1 CA109618(to B.L.) and RO1 GM053396 (to D.J.K.).

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