B. DEPARTMENT OF CELL GENETICS
B-a. Division of Cytogenetics - Tamotsu Yoshimori Group

RESEARCH ACTIVITIES

Tamotsu Yoshimori

--Most of membrane-bound organelles inside eukaryotic cells are linked each other by dynamic membrane trafficking regulated by a set of specific proteins. Membrane traffic is essential not only to survival of each cell but also to various functions organizing the multi-cellular system, e.g., formation of cell polarity and intercellular communication. We aim to unravel molecular mechanisms of membrane traffic and their roles in physiological functions and diseases in animals, which must produce knowledge contributing clinical medicine. We are now focusing on two trafficking routes; autophagy and the endosomal system. Autophagy is membrane traffic delivering cytoplasmic components to lysosomes for bulk degradation. The process is mediated by the formation of the double membrane-bound autophagosomes. Endosomes receive macromolecules taken up by endocytosis from outside. The cargo is then either sorted to lysosomes or recycled back to the plasma membrane.

(1) Three mammalian homologues of yeast Atg8 localize to autophagosomal membrane depending on post-translational processing

Yukiko Kabeya¹, Noboru Mizushima², Akitsugu Yamamoto³, Satsuki Oshitani-Okamoto¹, Yoshinori Ohsumi¹ and Tamotsu Yoshimori (¹National Institute for Basic Biology, ²Tokyo Metropolitan Institute of Medical Science, ³Nagahama Institute of Bio-Science and Technology)

--We previously reported that rat LC3, a homologue of yeast Atg8, localizes to autophagosomal membranes after post-translational modifications. The C-terminal fragment of LC3 is cleaved immediately following synthesis to yield a cytosolic form, LC3-I. A subpopulation of LC3-I is further converted to an autophagosome-associating form, LC3-II by the ubiquitylation-like conjugation system. In this study, we showed that [¹⁴C]-ethanolamine was preferentially incorporated into LC3-II, suggesting that LC3-II is a phosphatidylethanolamine (PE)-conjugated form like a membrane-binding form of yeast Atg8¹⁾. 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.
--There are at least two other homologues of yeast Atg8, γ-aminobutyric-acid-type-A-receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16kDa (GATE16) in mammals. We found that they also generate form II and its generation correlated with autophagosomal association of GABARAP and GATE16¹⁾. The results suggest that all mammalian Atg8 homologues receive a common modification to associate with autophagosomal membrane as the form II. GABARAP and GATE16 have been suggested to be involved in the GABA_A-receptor transport and an intra-Golgi transport, respectively. However, it remains a possibility that they participate in autophagy in addition to or instead of their functions originally described.

(2) Autophagy plays a pivotal role in survival during the early neonatal starvation period

Akiko Kuma¹, Masahiko Hatano², Makoto Matsui¹, Akitsugu Yamamoto³, Haruaki Nakaya², Tamotsu Yoshimori, Yoshinori Ohsumi⁴, Takeshi Tokuhisa² and Noboru Mizushima¹ (¹Tokyo Metropolitan Institute of Medical Science, ²Chiba University, ³Nagahama Institute of Bio-Science and Technology, ⁴National Institute for Basic Biology)

--At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. We showed that neonates adapt to this adverse circumstance by inducing autophagy to degrade cytoplasmic constituents within lysosomes⁶⁾. The level of autophagy in mice, which was monitored by GFP-LC3 expressed in the transgenic mice¹⁾, 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 U3085self' proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.

(3) Autophagic machinery provides a cellular defense system against invasion by group A Streptococcus

Ichiro Nakagawa¹, Takahiro Kamimoto, Akitsugu Yamamoto², Noboru Mizushima³, Kayoko Tsuda, Atsuki Nara, Junko Funao¹, Masanobu Nakata¹, Atsuo Amano¹, Shigeyuki Hamada¹ and Tamotsu Yoshimori (¹Osaka University, ²Nagahama Institute of Bio-Science and Technology, ²Tokyo Metropolitan Institute of Medical Science)

--A Gram-positive bacteria, group A Streptococcus (GAS) is the etiological agent for a diverse collection of human diseases. GAS invades non-phagocytic cells, but the destination of GAS after internalization is not well understood. We found that the autophagic machinery could effectively eliminate GAS within non-phagocytic cells⁷). GAS that had invaded HeLa cells or MEF was first trapped by endosomes and then successfully escaped to the cytoplasm using bacterial cytolysin SLO. Then, the cytoplasmic GAS was selectively sequestered by large, unique compartments bearing LC3, the formation of which was specifically induced by bacterial infection and required the Atg5 gene. The numbers of living GAS decreased to less than 10% at 4 h of post-infection and this reduction was completely suppressed in autophagy deficient Atg5^-/- cells. In these cells, GAS survived, multiplied, and were released from the cells. The compartments trapping GAS eventually fused with lysosomes, in which enzymes degraded and killed the GAS. The autophagic machinery could only recognize GAS that had escaped from endosomes to the cytoplasm. Based on these results, we concluded that the autophagic machinery can act as an innate defence system against invading pathogens.

(4) Intracellular Shigella escapes from Autophagy

Michinaga Ogawa¹, Tamotsu Yoshimori, Toshihiko Suzuki¹, Hiroshi Sagara¹, Noboru Mizushima² and Chihiro Sasakawa¹ (¹University of Tokyo, ²Tokyo Metropolitan Institute of Medical Science)

--Shigella is a group of Gram-negative bacteria causing Shigellosis. The invasion of Shigella into the colonic epithelium initiates the disease. IcsB, one of the Shigella flexneri effectors, is secreted via the type III secretion system of cytoplasmic bacteria and located on the bacterial surface. The icsB mutant is fully invasive, but defective in spreading within host cells. To clarify the role of IcsB in promoting infection, we investigated the intracellular behaviors of the icsB mutant⁸⁾. As a result, we found that the mutant bacteria were trapped by autophagy in the cytoplasm. IcsB did not directly inhibit autophagy. Rather, Shigella VirG, a protein required for intracellular actin-based motility, induced autophagy by binding to Atg5. In non-mutant Shigella, this binding is competitively inhibited by IcsB binding to VirG. Thus, in contrast to GAS, Shigella in the cytoplasm is able to escape from attack of autophagy by secreting IcsB which act as an inhibitor of recognition of the bacteria by Atg5.

(5) The intracellular inclusions containing mutant α₁-antitrypsin Z are propagated in the absence of autophagic activity

Takahiro Kamimoto, Shisako Shoji, Noboru Mizushima¹, Kyohei Umebayashi, Tunda Hidvegi², David H. Perlmutter² and Tamotsu Yoshimori (¹Tokyo Metropolitan Institute of Medical Science, ²University of Pittsburgh)

--Formation of intracellular inclusions comprised of terminally misfolded proteins is the most obvious hallmark of diseases that are collectively termed conformational disease. Mutant α₁-Antitrypsin Z (α₁-ATZ) protein accumulates within the endoplasmic reticulum (ER) of the liver cells as an aggregated polymer and is associated with the development of chronic liver injury and hepatocellular carcinoma in hereditary α₁-antitrypsin (α₁-ATZ) deficiency. Previous studies have suggested that efficient intracellular degradation of _α1-ATZ is correlated with protection from liver disease in α₁-ATZT deficiency, and that the ubiquitin proteasome system accounts for a major, but not sole, route of α₁-ATZ disposal. Yet autophagy has also been implicated in the pathophysiology of α₁-ATZ deficiency. In this study, to provide genetic evidence that autophagy can mediate disposal of α₁-ATZ, we used autophagy deficient Atg5-/- cells. The results showed that in the absence of autophagy, degradation of α₁-ATZ was retarded and that α₁-ATZ accumulated in characteristic cellular inclusions colocalized with calnexin and ubiquitin. These data provide definitive evidence that autophagy can participate in the quality control/degradative pathway for α₁-ATZ and suggest that autophagic degradation plays a fundamental role in preventing the formation of characteristic cytoplasmic inclusions.

(6) Analysis of invasion of Porphyromonas gingivalis into cells by using beads coated with the bacterial vesicles

Kayoko Tsuda, Ichiro Nakagawa¹, Kyohei Umebayashi, Atsuo Amano¹ and Tamotsu Yoshimori (¹Osaka University)

--Porphyromonas gingivalis, Gram-negative anaerobic bacterium, is considered to be a bona fide pathogen of adult periodontitis, which is the most prevalent chronic disease among human over the world. P. gingivalis can internalize into primary gingival epithelial cells and other cell types. To clarify the mechanisms underlying the invasion and itinerary of the pathogen within host cells, we have used fluorescent beads coated with vesicles secreted by P. gingivalis. The beads efficiently entered into cells through membrane trafficking, while the internalization did not occur at all when beads were coated with BSA or the heat-inactivated vesicles. We have been investigating cellular components involved in the beads internalization and fate of the internalized beads.

(7) Reconstitution of initiation of autophagosome formation by using semi-intact cells

Shunsuke Kimura, Atsuki Nara, Yoshitaka Nagai¹ and Tamotsu Yoshimori (¹Osaka University)

--In vitro reconstitution of membrane trafficking by using the semi-intact cell systems is a powerful tool to resolve its molecular machinery. To elucidate mechanisms underlying autophagic membrane dynamics, we established semi-intact cells by treatment of cultured cells with a bacterial toxin, Streptolysin O, which forms micro pores on the plasma membranes and allow us to access the cytoplasm directly. We succeeded to reconstitute formation of the small vesicle decorated with GFP-Atg5, which is a precursor of autophagosome in the semi-intact cells by adding the cytosol fraction and ATP-regenerating system. The cytosol isolated from starved cells was more effective than that from cells cultured in nutrient-rich condition. Since autophagy is known to be induced by starvation, the result indicates that the starved cell cytosol contains factor(s) triggering autophagosome formation. Moreover, we found that adding of the recombinant protein containing expanded polyglutamine fragment, which causes a class of inherited neurodegenerative diseases, so-called polyglutamine diseases, including Huntington's disease, induced autophagy in this system. We are screening the cellular molecules recognizing the starvation signal or the expanded polyglutamine fragment to induce autopagy.

(8) Ubiquitin-dependent sorting of the epidermal growth factor receptor in the endocytic pathway

Kyohei Umebayashi and Tamotsu Yoshimori

--When plasma membrane proteins are ubiquitinated, they follow the endocytic pathway to lysosomes. In the case of the epidermal growth factor receptor (EGFR), the Cbl E3 ligase is responsible for ligand-dependent ubiquitination. It has been considered that the ubiquitination of the receptor occurs in the plasma membrane, however, we found that Cbl is localized to early endosomes when cells are stimulated with EGF. Early endosomes are composed of distinct membrane domains, which may represent various transport directions from this organelle. Hrs sorts ubiquitinated cargoes to lysosomes, and is localized to specific domains of early endosomal membrane. The localization patterns of Cbl and Hrs were overlapped very well, suggesting that Cbl is localized to early endosomal subdomains where cargoes destined for lysosomes are concentrated. It is known that expression of a dominant negative form Cbl(C381A) abolishes the ubiquitination of EGFR. Strikingly, we found that Cbl(C381A) does not inhibit the internalization of EGF from the plasma membrane. EGF could reach endosomes where it was colocalized with Cbl(C381A). Thus, ubiquitination of EGFR is not obligatory for the internalization process. Both the ubiquitination and the ubiquitin-dependent sorting of EGFR may occur in early endosomal subdomains. Tracking the localization after EGF stimulation suggested that Cbl binds EGFR in the plasma membrane, remains associated in early endosomes, and then becomes separated from the receptor. We have obtained an implication that an AAA ATPase SKD1 regulates the ubiquitinated status of EGFR, and will investigate the molecular mechanisms in detail.

PUBLICATIONS

Papers
1. Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T. and Ohsumi, Y. (2004). In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell 15, 1101-1111.
2. Prentice, E.W., Jerome, G., Yoshimori, T., Mizushima, N. and Denison, M.R. (2004). Coronavirus Replication Complex Formation Utilizes Components of Cellular Autophagy. J. Biol. Chem. 279, 10136-10141.
3. Kabeya, Y., Mizushima, N., Oshitani-Okamoto, S., Ohsumi, Y. and Yoshimori, T. (2004). LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J. Cell Sci. 117, 2805-2812.
4. Fujita, H., Umezuki, Y., Imamura, K., Ishikawa, D., Uchimura, S., Nara, A., Yoshimori, T., Hayashizaki, Y., Kawai, J., Ishidoh, K., Tanaka, Y. and Himeno, M. (2004). Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B. J. Cell Sci. 117, 2997-3009.
5. Birkeland, H.C.G., Simonsen, A., Gillooly, D.J., Mizushima, N., Kuma, A., Yoshimori, T., Slagsvold, T., Brech, A. and Stenmark, H. (2004). Alfy, a novel FYVE domain-containing protein associated with protein granules and autophagic membranes. J. Cell. Sci. 117, 4239-4251.
6. Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., Ohsumi, Y., Tokuhisa, T. and Mizushima, N. (2004). Role of autophagy during the early neonatal starvation period. Nature 432, 1032-1036.
7. Nakagawa, I., Amano, A., Mizushima, N., Yamamoto, A., Yamaguchi, H., Kamimoto, T., Nara, A., Funao, J., Nakata, M., Tsuda, K., Hamada, S. and Yoshimori, T. (2004). Autophagy defenses cells against invading group A Streptococcus. Science 306, 1037-1040.
8. Ogawa, M., Yoshimori, T., Suzuki, T., Sagara, H., Mizushima, N. and Sasakawa, C. (2004). Escape of Intracellular Shigella from Autophagy. Science published online 2 December [DOI: 10.1126/science.1106036].
9. Nakatsukasa, K., Okada, S., Umebayashi, K., Fukuda, R., Nishikawa, S. and Endo, T. (2004). Roles of O-mannosylation of aberrant proteins in reduction of the load for endoplasmic reticulum chaperones in yeast. J. Biol. Chem. 279, 49762-49772.

Reviews
10. Yoshimori, T. (2004). Autophagy: a regulated bulk degradation process inside cells Biochem. Biophys. Res. Commun. 313, 453-458.
11. 吉森 保(2004)蛋白質大規模分解システムとしてのオートファジー:明らかになってきた多彩な生理機能.蛋白質核酸酵素増刊号「細胞における蛋白質の一生」49, 1029-1032.
12. 吉森 保(2004)オートファジーと疾患.医学のあゆみ211, 147-151.
13. 梅林恭平(2004)ステロールとユビキチン―ポストゴルジでのタンパク質選別輸送における役割―日本農芸化学会誌78, 39-41.

ORAL PRESENTATIONS

1. Yoshimori, T. New insights into molecular mechanisms and roles of autophagy. Symposium 7: Sorting and Selection in Membrane Traffiking, The 57^th Annual Meeting of Japan Society for Cell Biology, Osaka, May, 2004.
2. 吉森 保.オートファジーと疾患.第9回病態と治療におけるプロテアーゼとインヒビター研究会,ワークショップ「オートファジーのメカニズムとパソロジー」,名古屋市,2004年7月

POSTER PRESENTATIONS

1. Umebayashi, K. and Yoshimori, T. Cargo ubiquitination in endosomes implicated by localization of an E3 ligase. The 57^th Annual Meeting of Japan Society for Cell Biology, Osaka, May, 2004.
2. Yoshimori, T. New insights into molecular mechanisms of autophagy and its role in diseases. ELSO 2004 Conference, Nice, September, 2004.
3. Umebayashi, K. and Yoshimori, T. Cargo ubiquitination in endosomes implicated by localization of an E3 ligase. ELSO 2004 Conference, Nice, September, 2004.
4. Yoshimori, T. Autophagic machinery provides a cellular defense system against invasion by a pathogenic bacteria, group A streptococcus. The American Society for Cell Biology 44^rd Annual Meeting, Washington DC, December, 2003.

EDUCATION

1. Dr. T. Yoshimori gave a lecture at Division of Pharmaceutical Science, Graduate School of Natural Science and Technology, Kanazawa University, January, 2004 (in Japanese).
2. Dr. T. Yoshimori gave a lecture at Kakegawa-nishi High School, Shizuoka-ken, March, 2003 (in Japanese).
3. Dr. T. Yoshimori gave a lecture at Graduate School of Integrated Science, Yokohama City University, March, 2004 (in Japanese).
4. Dr. T. Yoshimori was invited to give a seminar at Institute for Frontier Medical Science,Kyoto University, March, 2004 (in Japanese).
5. Dr. T. Yoshimori gave a lecture at Graduate School of Pharmaceutical Science, The University of Tokyo, July, 2004 (in Japanese).
6. Dr. T. Yoshimori was invited to give a seminar at Geneva University Sciences II, Geneva, September, 2004.
7. Dr. T. Yoshimori was invited to give a seminar at The Institute of Medical Science, The University of Tokyo, September, 2004 (in Japanese).
8. Dr. K. Umebayashi was invited to give a seminar at the Norwegian Radium Hospital, Oslo, September, 2004.

SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. T. Yoshimori was appointed for a councillor of the Japan Society for Cell Biology.
2. Dr. T. Yoshimori was appointed for an associate editor of “Cell Structure and Function".
3. Dr. T. Yoshimori was appointed for a member of editorial board of “Autophagy".
4. Dr. T. Yoshimori organized a symposium “Sorting and Selection in Membrane Traffiking" at The 57^th Annual Meeting of Japan Society for Cell Biology, Osaka, May, 2004.