Critical Review. Protein Turnover. Yoshinori Ohsumi National Institute for Basic Biology, Okazaki, Japan INTRODUCTION

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1 IUBMB Life, 58(5 6): , May June 2006 Critical Review Protein Turnover Yoshinori Ohsumi National Institute for Basic Biology, Okazaki, Japan Summary The mechanisms and physiological meanings of protein turnover are crucial subjects in the understanding of life. However, for a long time, protein degradation was neglected by most biologists, and was thought of as a rather passive cellular process. IUBMB Life, 58: , 2006 Keywords Protein degradation; lysosomes; autophagy; ubiquitin/ proteasome. INTRODUCTION Every cellular activity is maintained by a balance between synthesis and degradation of cellular proteins. Nutritionists tell us that we need to eat grams of protein every day, as a source of building blocks of protein, amino acids. We synthesize about grams of protein every day. But we are able to survive for long time without any ingestion of protein, which does not mean a cessation of protein synthesis during starvation. These facts indicate that most proteins are synthesized from amino acids derived from degradation of pre-existing proteins, in other words by organisms capable of efficient recycling of proteins. LYSOSOMES More than 55 years ago C. de Duve discovered lysosomes by his cell fractionation procedures as an organelle containing various hydrolytic enzymes (1). It must be a reasonable strategy for cells to segregate the dangerous degradation process within the membrane of an organelle. Therefore, a basic problem of lysosomal degradation is how the substrates to be degraded become accessible to the lysosomal enzymes. Electron microscopic studies revealed the membrane dynamism of lysosomes and main delivery routes to the Received 12 April 2006; accepted 12 April 2006 Address correspondence to: Prof. Yoshinori Ohsumi, Division of Molecular Cell Biology, National Institute for Basic Biology, Myodaiji, Okazaki , Japan. yohsumi@nibb.ac.jp lysosome. One is the endocytic pathway (heterophagy), which degrades extracellular proteins, even bacteria and also plasma membrane proteins, via endocytic vesicles and endosomes (2). The other was called autophagy, which means self eating, the process of degradation of cytoplasmic proteins and organelles. A portion of cytoplasm including organelle is enclosed by a membrane sac, so called isolation membrane, and results in a double membrane organelle, autophagosome (3). Subsequently it fuses with lysosome, and the contents are digested and reutilized. Autophagy in mammals seems to be diverse in different organs and also on different physiological demands, and many schematic depictions of autophagy were proposed. The lysosome is so dynamic and heterogeneous that biochemical analyses on its dynamics have been limited. No good marker proteins for the autophagic degradation, and no genes involved in this process have been elucidated. Therefore, detection of autophagosomes and autolysosomes (autophagosomes fused with lysosome) in electron micrography was the only way to analyze autophagy. Not much progress has been made to dissect the mechanism of autophagy. TWO MAJOR SYSTEMS OF PROTEIN DEGRADATION For a long time it was assumed that most intracellular proteins are degraded in the lysosome. But about 20 years ago, researchers discovered that a small number of groups revealed an energy-dependent proteolysis in the cytosol. It opened up a novel vast field of the ubiquitin/proteasome system. It was proved that many critical cellular events are regulated by this system. Most people started to realize that protein degradation is as important as synthesis, and now it becomes clear that significant percentages of the genome are related to protein degradation, and proteolysis has become a hot subject in biology. Each protein has its own lifetime within a broad range from a few minutes to more than one month. It is obvious that cells require different pathways of protein turnover for different purposes. It is generally accepted that short-lived proteins, which consist of most regulatory proteins and damaged or misfolded proteins, are degraded by the ubiquitin/proteasome ISSN print/issn online Ó 2006 IUBMB DOI: /

2 364 OHSUMI system (4), while long-lived proteins are degraded in the lysosome via autophagy. Since our body is made up mostly of long-lived proteins, from the point of view of nutrition autophagy is crucial for life. The degradation by proteasome is held in the cytosol, the site of biosynthesis, therefore, requires a strict recognition of target proteins with consumption of energy of ATP hydrolysis. In contrast autophagy plays roles in a bulk and non-selective protein turnover. AUTOPHAGY IN YEAST In order to make a breakthrough in autophagy, a good model system was necessary. Budding yeast, S. cerevisiae, turned out to be the most useful organism for this purpose. Yeast cells have relatively large compartments, vacuoles, which are acidified by V-type ATPase and contain various kinds of hydrolytic enzymes. So it had been postulated that the vacuole is analogous to the lysosome in mammals. However, nothing was known about what kind of proteins and how proteins were degraded within this organelle. Many mutants of vacuolar functions show a sporulation-negative phenotype. The sporulation, meiosis of yeast, is triggered by depletion of nitrogen from the medium. For this cell differentiation, bulk protein degradation in the vacuole must be essential. I observed the vacuolar proteinase-deficient mutant cells under nitrogen starvation, expecting to identify the structures involved in delivery of proteins to the vacuoles, and found an obvious morphological change of the vacuoles. After 30 min of starvation spherical bodies appeared in the vacuoles, and moved around vigorously by Brownian motion. For up to 10 h these bodies gradually accumulated in the vacuoles and finally filled inside the vacuoles (5). Electron microscopy of the mutant cells under starvation revealed that these bodies in the vacuole, named autophagic bodies, are mostly single membrane-bound vesicles, containing ribosomes and other cellular structures including mitochondria and rer (5). Then double membrane structures of the same size to autophagic bodies, yeast autophagosomes, were found in the cytoplasm of the starved cells. Fusion images between the outer membrane of autophagosome and the vacuolar membrane were obtained by rapid freezing or freezefracture electron microscopy (6, 7). Autophagic bodies were nm in diameter, about 500 nm on average, which delivers about 0.2% of the cytoplasm in a quantized manner. Membrane dynamics of yeast autophagy is topologically the same as macroautophagy in mammals, though the vacuole is much larger than the lysosomes. A schematic drawing of autophagy in yeast is shown in Fig. 1. INDUCTION OF AUTOPHAGY In yeast, the extent of autophagy is negligibly small when growing in a rich medium. Similar membrane phenomena were induced by carbon, sulfate, phosphate and single auxotrophic amino acid starvation (5). These observations strongly indicate that yeast cells take up a portion of the cytoplasm to the lytic compartment via autophagosomes in adverse conditions for growth. So far there is no mutant unable to respond to a specific starvation signal. Autophagy should be primarily a physiological response to nutrient limitation and may be under the control of common but unknown starvation signals. High camp levesl blocked autophagy and activated A-kinase mutants do not induce autophagy (8), indicating that autophagy is regulated in an opposite manner to cell growth. When rapamycin, a specific inhibitor of the Tor kinase, is added to a nutrient-rich medium, yeast cells induce autophagy just like during nitrogen starvation (8). Thus the Tor kinase negatively regulates autophagy during growing conditions as a master regulator. At present a regulator of Tor and downstream pathway toward autophagy are not fully understood. The mechanism of signal transduction of autophagy is a still unanswered question. MEMBRANE DYNAMICS DURING AUTOPHAGY The most critical event in autophagy is not the proteolytic step, but the sequestration of a portion of cytoplasm to be degraded. This autophagosome formation is a unique membrane dynamic, different from the conventional membrane traffic. For a long time the origin of the autophagosome membrane was proposed to be the ER (9). We also showed that membrane flow from the ER is necessary for autophagy (10). Another group reported post-golgi and other organelle transport are involved in autophagsoome formation. The autophagosomal membrane in yeast has a distinct morphology; thinner than other organelle membranes and the outer and inner membranes stick together without lumenal space (5, 6). In freeze fracture images the inner membrane completely lacked intra-membrane particles, while the outer membrane contained sparse but significant particles (7), which may participate in targeting and fusion of the autophagosome to the vacuole. The inner and outer membranes are somehow differentiated and specialized for delivery of cytoplasm to the vacuole, though they must be derived from both sides of the isolation membrane. By electron microscopy we could detect a cup-shape intermediate membrane structure at low frequency, but so far nobody has shown membrane vesicles involved in the membrane elongation step of the isolation membrane. We proposed that autophagosome formation is not simple an enwrapping process by a pre-existing large membrane structure such as the ER, but rather de novo assembly of a new membrane from its constituents (10, 11). Many questions still remain to be answered.: Where do the constituents of the membrane originate? How is the isolation membrane organized? What factors are involved in the morphogenesis of the isolation membrane? How does it seal to form a closed double-membrane-bound compartment? What is the fusion machinery of autophagosome to the vacuole?

3 PROTEIN TURNOVER 365 Figure 1. A schematic drawing of autophagy in yeast. GENETIC APPROACH TO THE AUTOPHAGY To elucidate the molecular mechanism of autophagy, we applied a genetic approach. The most characteristic feature of yeast autophagy is that we are able to monitor the progress of autophagy under a light microscope as the accumulation of autophagic bodies. Taking advantage of this, we attempted to obtain autophagy-defective mutants. Mutagenized vacuolar proteinase-deficient cells that failed to accumulate autophagic bodies during starvation were selected under a light microscope and a mutant, apg1 was obtained (12). The apg1 mutant did not induce bulk protein degradation under starvation, and homozygous apg1/apg1 diploid cells did not sporulate. The apg1 mutant grew normally in a rich medium, but could not maintain viability under long nitrogen starvation. To obtain more apg mutants, this loss of viability on starvation was used for the first screening, assuming that it is due to the defect in autophagy. About 100 autophagy-defective mutants were isolated, and divided into 14 groups (apg2-apg15) by complementation analysis (12). The original strategy for isolation of autophagy-defective mutants was quite efficient, so since then only two genes are added as typical autophagy mutants using two hybrid screens with known Apg proteins. Another approach taken by Thumm and his colleagues was immuno-screening of cells that retain a cytosolic enzyme, fatty acid synthase, after starvation. By this method originally 6 aut mutants were obtained (13). Klionsky s group has been studying the pathway of maturation of aminopeptidase I (API), one of the vacuolar enzymes, and isolated defective mutants in the process (14). Unlike other vacuolar enzymes, the proform of API is transported from the cytosol to the vacuole via the Cvt pathway (15). The cvt mutants defective in the Cvt pathway significantly overlapped with autophagy defective apg and aut mutants (16, 17), though the two pathways are apparently different; one is degradative and starvation-induced, and the other is biosynthetic and constitutive. EM analyses of the Cvt pathway clearly showed that the Cvt pathway is mediated by quite similar membrane dynamics to autophagy (18, 19). Later, several groups

4 366 OHSUMI identified autophagy-related genes in S. cerevisiae and other yeast species, and named them differently. To avoid confusion, recently all groups involved agreed to use a novel nomenclature for the autophagy-related gene, ATG. The original APGx is now renamed as ATGx (20). Among ATG genes, the original APG genes and ATG18, and ATG29 are involved in the autophagosome formation, so here, APG will be used for these genes collectively. CHARACTERIZATION OF AUTOPHAGY-DEFECTIVE MUTANTS The apg mutants fail to induce bulk protein degradation under nutrient-depleted conditions, indicating that autophagy is the major pathway of bulk protein degradation. They grow normally and show no defects in stress responses, vacuolar functions, secretion, and endocytosis, indicating that autophagy is not essential for vegetative growth in yeast (12). Autophagy-defective mutants start to die after 2 days of starvation and almost completely lose viability after 1 week (12). Homozygous diploids with any apg mutation cannot sporulate, indicating that cell remodeling must require bulk protein degradation via autophagy (12). Recently we examined the amino acid pool during nitrogen starvation. In wild type cells, amino acid levels drop dramatically for the first 3 h of starvation, but recover and maintain a certain level; in contrast, in atg mutants most amino acid levels decrease further and some become very low (21). The amount of nitrogen-starvation-inducible proteins was severely reduced in atg mutants; probably protein synthesis is limited by the shortage of amino acids. Recycling of protein via autophagy must be essential for survival during starvation. All apg mutants did not accumulate autophagosomes in the cytoplasm during starvation, indicating that thess genes have functions at or before the formation step of the autophagosome, and further studies on the Apg proteins confirmed that all these proteins function at the autophagosome formation step. FURTHER GENES REQUIRED FOR AUTOPHAGY Typical autophagy mutants like the original apg mutants seem to be nearly saturated. However, the strategies of screen eliminated mutants of aberrant vacuole morphology, partially defective mutants, and of genes sharing with other essential functions. Now it is obvious that normal autophagy requires more both known and unknown genes. Most Gcn proteins are necessary for the normal extent of autophagy. Several early secretory genes such as SEC12 an SEC24 were shown to be required for autophagy (10). Several mutants such as vam5 and ypt7 show accumulation of autophagosomes in the cytoplasm under starvation, suggesting the fusion machinery of autophagosome with the vacuole shares SNARE molecules with that for vacuolar homotypic fusion. In wild type cells autophagic bodies effectively disappear within a minute. Atg15 and Atg22 were shown to be involved in this process (22, 23). Atg15 contains a putative lipase domain, but its activity has not yet been proved. Acidification of the vacuole is a requisite for effective digestion of autophagic bodies, since a defect in every subunit of V-ATPase (Vma) causes an accumulation of autophagic bodies in the vacuole (24). It is still an open question why the autophagic body membrane is immediately disintegrated in less than a minute in the vacuoles. FUNCTION OF ATG PROTEINS Almost all ATG genes were unknown genes, except Atg6/ Vps30, which is required for vacuolar protein sorting (25). Even in yeast, autophagy genes had been overlooked for a long time because they exhibit phenotypes only under starvation conditions. Recent analyses revealed that Atg proteins are classified into several functional units. One of the most remarkable findings is the discovery of two ubiquitin-like conjugation systems in Atg proteins (26, 27). Actually half of the APG genes are involved in these novel conjugation systems. A brief summary of our present knowledge of the Atg protein system follows. (A) Atg12 Protein Conjugation System Apg12 is a novel ubiquitin-like protein (UBL) (26). Atg12 is activated by an activating enzyme (E1), Atg7, and then transferred to a conjugating enzyme (E2), Atg10, by forming thioester conjugates, and finally an isopeptide conjugates with Atg5. The Atg12 conjugation reaction is similar to ubiquitination, but has distinct features. Atg12 is synthesized as an active form with a single glycine at the C-terminus. Recently the crystal structure of Arabidpsis Atg12 was solved in Dr Inagaki s lab, showing a ubiquitin-fold at the C-terminal region. A C-terminal ubiquitin fold is necessary and sufficient for conjugation and also for autophagy. Atg5 is the only target molecule of the Atg12 modification. Atg12 and Atg5 form a conjugate immediately after their synthesis and free forms are hardly detectable in cell lysates. This conjugation reaction is irreversible, since deconjugating enzymes have not been found. Conjugate formation is not affected by autophagy-inducing conditions and the conjugate behaves just like a single polypeptide and functions as a part of the machinery of autophagosome formation. The conjugate further forms a complex with Atg16. Apg16 binds to Atg5 at the N-terminal region and forms an homo-oligomer through a coiled-coil region. Atg12- Atg5-Atg16 forms a large complex of about 350 kda, which is essential for autophagy (28). In mammals this complex is shown to reside on the outer surface of the isolation membrane, but dissociates from the membrane when autophagosome has been completed. (B) Atg8 Lipidation System Atg8 is another UBL and has many homologues in eukaryotes. Immunoelectron microscopy showed that Atg8

5 PROTEIN TURNOVER 367 is localized on the isolation membrane, autophagosome and autophagoc body during autophagosome formation (29). The C-terminal arginine is processed by Atg4 (Atg8DR), a novel and well conserved cysteine proteinase family protein (30). Then Atg8 is activated by Atg7, and then is transferred to Atg3, an E2 enzyme. Atg7 is a unique E1 enzyme which activates two different UBLs, Atg12 and Atg8, and transfers them to each E2 molecule, Atg10 and Atg3, respectively (27). By SDS-PAGE in the presence of 6 M urea two forms of Atg8 were separable (30). Mass spectrography of the modified form of Atg8 showed a covalent conjugate of Atg8 to phosphatidylethanolamine (PE). Importantly Atg8-PE formation was reversible and the processing enzyme, Atg4, functions also for this deconjugation (27, 30). The expression of Atg8 is induced by nitrogen starvation, and the Atg8-PE level is also elevated (29). The cycle of Atg8-lipidation reaction is necessary for normal autophagy (30). Atg12 and Atg8 conjugation systems work concertedly; they not only share the same E1 enzyme, Atg7, but also functionally, because the Atg8-PE level is severely reduced in Atg12 system components, Atg5, Atg10, and Atg12 in mutants (31). Recently we succeeded to reconstitute the in vitro lipidation reaction using purified Atg8DR, Atg7, Atg3, and PE-containing liposome (32). This system will elucidate the molecular details of this interesting reaction. (C) Atg1 Kinase Complex The third protein complex required for autophagy is the Atg1 kinase complex. Atg1 possesses a serine/threonine kinase domain at the N-terminus region. Kinase-negative Atg1 mutant could not induce autophagy, implying that kinase activity is essential for function (33, 34). Atg1 kinase activity is enhanced during induction of autophagy, which is important for the regulation of autophagosome formation (35). Atg1 physically interacts with Atg13, Atg17 and Atg11/ Cvt9. Atg13 is a highly phosphorylated protein under nutrient-rich condition. On starvation or addition of rapamycin, it is immediately dephosphorylated by an as yet unknown phosphatase (34). Reversely, on addition of nutrients to starved cells, Atg13 is rapidly hyperphosphorylated. The phosphorylation state of Atg13 is controlled by the nutrient conditions through the Tor signaling pathway. Under starvation, Atg13 is tightly associated with Atg1, while under nutrient rich conditions the affinity is lowered (34). Atg17 binds to Atg13 and forms a ternary complex with Atg1, which causes activation of Atg1 kinase activity. Atg17 exists as a large complex. (D) Autophagy Specific PI3 Kinase Complex The fourth complex is an autophagy-specific PI3 kinase complex. ATG6 is allelic to VPS30 (25). Vps30/Atg6 has a dual function for vacuolar protein sorting and autophagy. There are at least two PI3 kinase complexes in yeast, Complex I and Complex II (36). Vps34, Vsp15 and Vps6 are common, but Atg14 and Vps38 define the specificity of the complex. Atg14, specifically required for autophagy, binds to Vps30 and Vps34 at the coiled-coil region. Vps34 is the sole phosphatidyl inositol 3-kinase in yeast and Vps15 is a regulatory protein kinase of Vps30. Atg14 determines the precise localization of PI3 kinase at the site of autophagosome formation, PAS (37). (E) Other Atg Proteins The remaining three Atg proteins, Atg2, Atg18 (Aut10) form a complex and weakly with Atg9. Atg9 is a putative multi-membrane spanning protein, but its localization does not fit to any known organelle marker (38). Their precise roles in autophagy are not known yet at the molecular level. However, they may play important roles in linkage between the above four reactions. SITE OF ATG PROTEIN FUNCTIONING PREAUTOPHAGOSOMAL STRUCTURE All Atg proteins function spatiotemporally in a very close manner during the autophagosome formation step. Recently we found that the autophagosome marker, Atg8, is localized in a small area close to the vacuole, and showed that almost all Atg proteins are localized also at the structure, the preautophagosome structure (PAS) (35). Temperature sensitive Atg1 ts mutant cells expressing GFP-Atg8 were completely defective in autophagosome formation; instead they showed a bright PAS at a restrictive temperature. On shiftdown to the permissive temperature, immediately, less brightly fluorescent structures, presumably autophagosomes, were generated from the PAS, and fused to the vacuole; consequently the vacuolar lumen stained brightly. This structure seems to be an organizing center of the autophagosome (35). Recently we performed systematic analyses of the PAS localization of every Atg protein in all atg disruptants (K. Suzuki, Y. Kubota, Y. Ohsumi, manuscript submitted). From this analysis we obtained a hierarchy map of Atg proteins. There are epistatic relations between functional units of Atg genes with PAS organization. The Atg12 system and lipidation are necessary for Atg8 to associate to the PAS. The Atg1 kinase complex is not required for the recruitment of Atg12 and Atg8. The PI3 kinase and Atg9 are necessary for the association of two conjugates to the PAS. Atg17 and Atg11 may function as scaffold proteins for all Atg protein localization to the PAS. Biochemical and fine structural investigation of the PAS will elucidate the molecular details of autophagsosme formation. ATG PROTEINS IN HIGHER EUKARYOTES Most of the ATG genes are conserved from yeast to mammals and plants, indicating that eukaryotic cells acquired the autophagic system at an early stage of evolution. Especially, two conjugation reactions are well conserved. Still several Atg proteins have not been identified in mammals or

6 368 OHSUMI plants. A requirement of PI3 kinase activity for autophagy is also reported in mammals, but the autophagy specific component, Atg14, has not been found. Possibly, as in the case of Atg16, sequence homology may not be sufficient to find the counterparts, or they may be yeast-specific factors. Just as in yeast, two conjugation systems provided a good marker for the membrane structures involved in autophagy in yeast. LC3, an Atg8 homologue, is also modified to LC3II, in a tightly membrane bound form after processing by Atg4 homologues. LC3 stains isolation membrane and autophagosomes. The Atg12 system is also well conserved, and Atg5 is localized in the isolation membrane, but dissociates when the autophagosome is completed, providing a good marker for the intermediate membrane. In multicellular organisms, autophagy should be diverse and regulated in a different manner. Our recent studies have made several marker proteins for autophagosomes available. To understand where and when autophagy occurs in vivo, transgenic mice systemically expressing GFP fused to LC3 were established. Cryosections of various organs were prepared and the occurrence of autophagy was examined by fluorescence microscopy. Active autophagy was observed in various tissues, such as the skeletal muscle, liver, heart, exocrine glands, thymic epithelial cells, lens epithelial cells and podocytes. The patterns of induction of autophagy in different tissues are clearly distinct. In some tissues, autophagy even occurs constitutively. This transgenic mouse is a useful tool to study mammalian autophagy (39). Elucidation of genes essential for autophagy in yeast facilitated the study of autophagy in various organisms Dictiosteirum discoidum, Caenorhabditis elegans, Drosophila, Arabidopsis and mouse and human. Knockout of Atg genes showed clearly the physiological significance of autophagy. 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