Endothelial Nitric-oxide Synthase Antisense (NOS3AS) Gene Encodes an Autophagy-related Protein (APG9-like2) Highly Expressed in Trophoblast

Abstract

Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles that is conserved in all eukaryotic species. The precise role of autophagy in mammalian development and potential involvement in disease remain to be discerned. Yeast Atg9p is the first integral membrane protein shown to be essential for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas its mammalian functional orthologue has yet to be identified. We have identified two human genes homologous to yeast Atg9p and designated these as APG9L1 and APG9L2. We have previously identified APG9L2 as NOS3AS, which participates in the post-transcriptional regulation of the endothelial nitric-oxide synthase (NOS3) gene on chromosome 7 through its antisense overlap. In human adult tissues, APG9L1 was ubiquitously expressed, whereas APG9L2 was highly expressed in placenta (trophoblast cells) and pituitary gland. In transient transfection assays we found that both proteins were primarily localized to the perinuclear region and also scattered throughout the cytosol as dots, a subset of which colocalized with an autophagosome-specific marker LC3 under starvation conditions. Finally, by the small interfering RNA-mediated knockdown of APG9L1 in HeLa cells, we demonstrated that APG9L1 is essential for starvation-induced autophagosome formation. In addition, APG9L2 can functionally complement APG9L1 in this process. These results, taken together with those of phylogenetic and sequence analyses, suggest that both APG9L1 and APG9L2 are functionally orthologous to the yATG9 in autophagosome formation. Moreover, APG9L2 is a vertebrate-specific gene that may have gained critical roles in mammalian-specific developmental events, such as placentation, through rapid evolution. Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles that is conserved in all eukaryotic species. The precise role of autophagy in mammalian development and potential involvement in disease remain to be discerned. Yeast Atg9p is the first integral membrane protein shown to be essential for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas its mammalian functional orthologue has yet to be identified. We have identified two human genes homologous to yeast Atg9p and designated these as APG9L1 and APG9L2. We have previously identified APG9L2 as NOS3AS, which participates in the post-transcriptional regulation of the endothelial nitric-oxide synthase (NOS3) gene on chromosome 7 through its antisense overlap. In human adult tissues, APG9L1 was ubiquitously expressed, whereas APG9L2 was highly expressed in placenta (trophoblast cells) and pituitary gland. In transient transfection assays we found that both proteins were primarily localized to the perinuclear region and also scattered throughout the cytosol as dots, a subset of which colocalized with an autophagosome-specific marker LC3 under starvation conditions. Finally, by the small interfering RNA-mediated knockdown of APG9L1 in HeLa cells, we demonstrated that APG9L1 is essential for starvation-induced autophagosome formation. In addition, APG9L2 can functionally complement APG9L1 in this process. These results, taken together with those of phylogenetic and sequence analyses, suggest that both APG9L1 and APG9L2 are functionally orthologous to the yATG9 in autophagosome formation. Moreover, APG9L2 is a vertebrate-specific gene that may have gained critical roles in mammalian-specific developmental events, such as placentation, through rapid evolution. Macroautophagy (or simply autophagy) is an intracellular degradation system used to recycle the majority of proteins and some organelles (1Kim J. Klionsky D.J. Annu. Rev. Biochem. 2000; 69: 303-342Crossref PubMed Scopus (320) Google Scholar, 2Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2968) Google Scholar, 3Mizushima N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (755) Google Scholar). In yeast, the autophagic pathway begins with the nonselective sequestering of cytoplasm into a double-membrane vesicle called the autophagosome. The outer membrane of the autophagosome is then targeted to the vacuole, where it fuses with the vacuolar membrane and releases the inner membrane with its contents (the autophagic body) into the vacuole lumen. Inside the vacuole, a variety of hydrolases breakdown the vesicle and degrade the cytoplasmic material (1Kim J. Klionsky D.J. Annu. Rev. Biochem. 2000; 69: 303-342Crossref PubMed Scopus (320) Google Scholar). The molecular mechanism of autophagy has been extensively studied through genetic screening and characterization of yeast genes required for autophagosome formation. Many of the 27 yeast autophagy-related genes identified so far are found to have homologues in higher eukaryotes, including mammalian species (4Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev. Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1015) Google Scholar). Although studies of such homologues at the cellular level have revealed the conservation of autophagosome formation mechanisms from yeast to mammalian species, the roles of autophagy at the organismal level have just begun to be understood. Autophagy is suggested to be involved in development, differentiation, growth regulation, and tissue remodeling in multicellular organisms (5Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3186) Google Scholar). Studies using mutants disrupted in autophagy genes have demonstrated a variety of phenotypes such as defective fruiting body formation in Dictyostelium discoideum (6Otto G.P. Wu M.Y. Kazgan N. Anderson O.R. Kessin R.H. J. Biol. Chem. 2003; 278: 17636-17645Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), premature death from the third larval to pupal stages in Drosophila melanogaster (7Scott R.C. Schuldiner O. Neufeld T.P. Dev. Cell. 2004; 7: 167-178Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar), abnormal dauer formation and short lifespan in Caenorhabditis elegans (8Melendez A. Talloczy Z. Seaman M. Eskelinen E.L. Hall D.H. Levine B. Science. 2003; 301: 1387-1391Crossref PubMed Scopus (1026) Google Scholar), and accelerated senescence in Arabidopsis thaliana (9Doelling J.H. Walker J.M. Friedman E.M. Thompson A.R. Vierstra R.D. J. Biol. Chem. 2002; 277: 33105-33114Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 10Hanaoka H. Noda T. Shirano Y. Kato T. Hayashi H. Shibata D. Tabata S. Ohsumi Y. Plant Physiol. 2002; 129: 1181-1193Crossref PubMed Scopus (462) Google Scholar). Recent generation of transgenic and knock-out mouse models for autophagy genes has provided new knowledge about the roles of autophagy in mammalian development and its possible links with other physiological events such as apoptosis. Analysis of transgenic mice expressing a fluorescent autophagosome marker (GFP 1The abbreviations used are: GFP, green fluorescent protein; EGFP, enhanced GFP; EYFP, enhanced yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein; RT, reverse transcription; ORF, open reading frame; siRNA, small interfering RNA; APG9, autophagy-related 9; y-, yeast; h-, human; m-, mouse. 1The abbreviations used are: GFP, green fluorescent protein; EGFP, enhanced GFP; EYFP, enhanced yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein; RT, reverse transcription; ORF, open reading frame; siRNA, small interfering RNA; APG9, autophagy-related 9; y-, yeast; h-, human; m-, mouse.-LC3) revealed that autophagy is induced in almost all tissues by nutrient starvation but with different patterns of response and that autophagy occurs actively without starvation treatments in some tissues (11Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1918) Google Scholar). Homozygous knock-out mice for Atg6/Becn1 are lethal at embryonic day 7.5, and heterozygous mice exhibit increased tumorigenesis (12Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. Rosen J. Eskelinen E.L. Mizushima N. Ohsumi Y. Cattoretti G. Levine B. J. Clin. Investig. 2003; 112: 1809-1820Crossref PubMed Scopus (1861) Google Scholar). Conversely, mice deficient for Atg5, which is shown to be essential and solely responsible for autophagosome formation (13Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1158) Google Scholar), appear almost normal at birth but die within 1 day of delivery due to energy depletion (14Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2385) Google Scholar). This suggests autophagy is seemingly dispensable for mouse early developmental events. In humans, mostly from morphological studies, possible roles of autophagy have been suggested in several disease conditions such as cancer, liver disease, muscular disorders, neurodegeneration, and pathogen infection (15Shintani T. Klionsky D.J. Science. 2004; 306: 990-995Crossref PubMed Scopus (2163) Google Scholar, 16Nakagawa I. Amano A. Mizushima N. Yamamoto A. Yamaguchi H. Kamimoto T. Nara A. Funao J. Nakata M. Tsuda K. Hamada S. Yoshimori T. Science. 2004; 306: 1037-1040Crossref PubMed Scopus (937) Google Scholar). However, whether autophagy might protect from or cause disease is unclear for the majority of cases. In plant and mammalian species, several autophagy genes such as APG1, APG4, and APG8 are shown to have paralogous gene copies (10Hanaoka H. Noda T. Shirano Y. Kato T. Hayashi H. Shibata D. Tabata S. Ohsumi Y. Plant Physiol. 2002; 129: 1181-1193Crossref PubMed Scopus (462) Google Scholar, 17Marino G. Uria J.A. Puente X.S. Quesada V. Bordallo J. Lopez-Otin C. J. Biol. Chem. 2003; 278: 3671-3678Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 18Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1110) Google Scholar). Although it remains to be determined whether all or a part of these genes function in autophagosome formation, some of those have been demonstrated to show tissue-specific differential expression patterns (10Hanaoka H. Noda T. Shirano Y. Kato T. Hayashi H. Shibata D. Tabata S. Ohsumi Y. Plant Physiol. 2002; 129: 1181-1193Crossref PubMed Scopus (462) Google Scholar, 17Marino G. Uria J.A. Puente X.S. Quesada V. Bordallo J. Lopez-Otin C. J. Biol. Chem. 2003; 278: 3671-3678Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), which may contribute to the developmental regulation of autophagic activity. Further characterization of the paralogous members would provide clues about how autophagy is involved in mammalian development, homeostasis, and possibly disease. We have previously reported that NOS3AS overlaps with NOS3 at chromosome 7q36 (19Scherer S.W. Cheung J. MacDonald J.R. Osborne L.R. Nakabayashi K. Herbrick J.A. Carson A.R. Parker-Katiraee L. Skaug J. Khaja R. Zhang J. Hudek A.K. Li M. Haddad M. Duggan G.E. Fernandez B.A. Kanematsu E. Gentles S. Christopoulos C.C. Choufani S. Kwasnicka D. Zheng X.H. Lai Z. Nusskern D. Zhang Q. Gu Z. Lu F. Zeesman S. Nowaczyk M.J. Teshima I. Chitayat D. Shuman C. Weksberg R. N. Friedman J.M. F. E. B. C.C. H. E. A. B.A. J. L. X. S. G.E. E. B. E. H. K. J.M. Li Science. 2003; PubMed Scopus Google and that it is involved in its post-transcriptional regulation Carson A.R. S. T. S.W. Nakabayashi K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The NOS3 has been suggested to be with a of including which is by and R. C. S. A. Walker W.A. D.J. J.M. F. J. Full Text PDF PubMed Scopus Google Scholar, G. J.A. M. Y. H. PubMed Scopus Google Scholar). However, the was in different R. S. T. H. A. B. S. D. A. J.R. K. Mol. PubMed Scopus Google Scholar, I. G. S. G. C. D. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. G. M. PubMed Scopus Google Scholar), and the role of NOS3 in the of remains a functional at the NOS3 highly expressed in we potential involvement of NOS3AS in the of We have revealed that NOS3AS a protein sequence with the yeast protein and that the human two homologues of the yeast APG9L1 on and APG9L2 on 7q36 Carson A.R. S. T. S.W. Nakabayashi K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). is the first integral membrane protein shown to be essential for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy T. J. M. C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar), whereas its mammalian functional orthologue has yet to be identified. In yeast, to but is found on T. J. M. C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar), that it is the autophagosome process. has also been shown to to other T. J. M. C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar, F. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). has been that and these other F. Klionsky D.J. Dev. Cell. 2004; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). In this as a first in the potential roles of APG9L2 and autophagy in development and disease, we determined APG9L2 in mammalian suggest that both APG9L1 and APG9L2 are functionally orthologous to the yeast gene in the cellular autophagosome formation. is a gene that may have gained critical roles in mammalian-specific for the and APG9L1 and APG9L2 sequence for the human APG9L2 gene was from two and and and identified by using and by The for the mouse and genes and were by the of the mouse sequence with the human and by of yeast Atg9p sequence was the protein using and to the Caenorhabditis A. thaliana C. elegans APG9L1 D. melanogaster APG9L1 The other homologues from and were from from sequence (the protein are gene models by and by the sequence conservation the human APG9L1 or APG9L2 sequence and the sequence of species from the at were used for the sequence of of yATG9 homologues were into proteins using PubMed Scopus Google was then used to all of the identified protein This protein with the was used to a sequence that the reading for all the This was then used to the phylogenetic in of were used for reverse with using The conditions were used for at for by of at for at for and at for The used for human genes are and for APG9L1 and for APG9L2 and and for sequence system was used with The used and are and for and for and for and and for the human A. conditions were for for and of for and for The were in for The from in two were used to the or expression level of APG9L1 and APG9L2 The was using a on the of a of The was to the using and as of were as previously K. K. J.R. PubMed Scopus Google Scholar). normal human placenta was was in and with and to were from the by through a The of was using 7 and were found to be subset of was in with and for at this and a for APG9L1 and APG9L2 was by using and The was as by The used are and and and and in the used for are was with using mouse placenta as The was with and and in and to that the mouse APG9L1 protein with a fluorescent protein or at the of APG9L1 and The were in the to for and and were in with growth at in and expressing EGFP, or with LC3 Y. Mizushima N. T. Yamamoto A. T. Noda T. E. Ohsumi Y. Yoshimori T. J. 2000; PubMed Scopus Google were and in with growth and were with expression using to autophagy by nutrient starvation were with and in for or and at of targeted to the human APG9L1 sequence was by The or was in the HeLa expressing using the The with from the and with were used to transfection of APG9L2 in the APG9L1 and APG9L2 genes and a higher level of sequence with the yeast in the APG9L1 protein with the APG9L2 protein that a a higher level of sequence The the proteins are the and the However, the and of the are conserved these Moreover, the and in and are conserved with the and of sequence the of a sequence at the of of but in or and yeast of of the are of of and of of the are of of and of The of the are of of and of in a new the of genes through we and protein from a of from mammalian species to eukaryotes, using the We then used S. K. M. 2001; PubMed Scopus Google to a phylogenetic from the sequence using a This that a vertebrate-specific the of This has to the of two in all including and mammalian have a was reported the human and mouse APG9L2 homologues with NOS3 in a Carson A.R. S. T. S.W. Nakabayashi K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). This to be conserved in all and in Although we were to this in and due to the of sequence that APG9L2 homologues are to NOS3 homologues in the mammalian APG9L2 genes appear to have accelerated by the in In the APG9L2 proteins are to the mammalian APG9L1 homologues to mammalian APG9L2 the the the mammalian is also the the and for and it that the mammalian homologues of the APG9L2 gene have for accelerated evolution. This rapid of the mammalian may have to the of mammalian-specific that be in critical developmental events such as protein sequence mammalian and in a new of APG9L1 and APG9L2 and on human adult tissues to the expression patterns of APG9L1 and and found that APG9L1 is expressed expression to be to placenta and pituitary with in and and This was also for the mouse and genes in the on the adult was found to be expressed at embryonic stages the adult this was to APG9L2 has role in development and disease. we to the expression of APG9L1 and APG9L2 in human from a and human of HeLa and with its expression in human and mouse tissues, APG9L1 was found to be expressed in all and with However, APG9L2 was found to be in cells, highly expressed in and cells, expressed in at a but expressed in the other the from to cells, APG9L2 a in its expression whereas APG9L1 a that APG9L2 expression is tissue-specific but also possibly of the cellular of the two mammalian proteins and possible functional we in by transient transfection of a fluorescent mouse or protein or be that with on its was previously shown to be functional T. J. M. C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar). or was with a marker into and the targeting sequence of the human with of expressed fluorescent proteins was under normal and starvation conditions. We that of starvation induced autophagosome formation in cells, a starvation was required for HeLa and we death of HeLa and of starvation we used of to autophagy in these and primarily localized at a perinuclear region and also localized throughout the cytoplasm under both normal and starvation conditions and The of the perinuclear and cytoplasmic within the However, of colocalized with We also expressed the sequence of with and found the protein with a sequence at the of APG9L2 but in we to the two proteins in patterns with to in conditions. However, this is the of of sequence are O. G. G. Cell Biol. 2001; PubMed Google Scholar). studies the of proteins to the functional of the of with an protein is involved in autophagosome formation in mammalian cells, we the of these proteins with that of which to the membrane throughout the of membrane and remains on the membrane the of autophagosome formation N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (755) Google Scholar). or was expressed in the expressing a fluorescent HeLa with with and with patterns were under starvation conditions. and were found to be with LC3 to a in all and This is to that of which is shown to both to and to other T. J. M. C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar, F. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). APG9L1 or APG9L2 are in the protein with LC3 are we of these fluorescent We that a of colocalized in these suggest the that both APG9L1 and APG9L2 proteins are involved in autophagosome formation. in whether proteins are required for mammalian autophagosome formation, we functional assays using the yeast in which autophagic can be by assays T. A. Y. Ohsumi Y. Biochem. PubMed Scopus Google Scholar). of mouse the function of yATG9 with the on Arabidopsis (10Hanaoka H. Noda T. Shirano Y. Kato T. Hayashi H. Shibata D. Tabata S. Ohsumi Y. Plant Physiol. 2002; 129: 1181-1193Crossref PubMed Scopus (462) Google Scholar). and plant proteins are to be from the to complement the yeast are the function of we APG9L1 expression in HeLa using We HeLa that APG9L1 but APG9L2 is expressed in this HeLa expressing were with targeted or for in the normal or the starvation for and used to the of on the APG9L1 expression level and autophagosome formation. The transfection of in conditions was targeted to APG9L1 was found to cause an in its expression level with the autophagosome formation was found to be by with the and we expression the of autophagosome formation by knockdown of and the for were in the HeLa expressing In the starvation higher of were in that is functional in autophagosome formation. The of the of in and were and be that transient expression of fluorescent as was in a small of the through the in this as shown in (the whereas the transfection of that APG9L1 is and APG9L2 is to APG9L1 in mammalian APG9L1 and APG9L2 to the Yeast have identified two mammalian genes and have shown that both proteins are involved in autophagosome formation. This is the first of the essential role of genes in mammalian APG9L1 is ubiquitously expressed and conserved from yeast to mammalian species, whereas APG9L2 is expressed in adult tissues and in species to but in eukaryotic species. These suggest that APG9L2 may contribute to tissue-specific and developmental of autophagy, and that it may have of mechanisms of cellular autophagosome formation are shown to be conserved from yeast to mammalian species, autophagy mechanisms at the organismal level are understood. multicellular organisms to have gained for autophagy genes through and such have to different tissue-specific expression patterns and possibly in the autophagy genes found to have in all but were found to have in A. APG9L2 to be the gene gained through evolution. The rapid of APG9L2 the mammalian by phylogenetic suggests that the gene might have gained mammalian-specific that are possibly involved in In some autophagy genes have through that may or may be involved in autophagy its function is the mammalian gene has a that is in its yeast orthologue N. A. Kobayashi Y. Yamamoto A. M. T. T. Ohsumi Y. Yoshimori T. J. Cell Sci. 2003; PubMed Scopus Google Scholar). is which is shown to be required for both autophagy and from X.H. S. Seaman M. K. B. Hibshoosh H. Levine B. Nature. PubMed Scopus Google Scholar, Z. S. C. Levine N. Sci. S. A. 2003; PubMed Scopus Google Scholar). Although we APG9L1 and APG9L2 in the it is possible that APG9L2 has vertebrate-specific of that the APG9L2 protein might be involved in the autophagic degradation of a sequence was at the of mammalian APG9L2 proteins but in the APG9L1 However, under the conditions in we of with a marker an in the patterns and is shown to to the and to small in the cytosol and is to these two F. Klionsky D.J. Dev. Cell. 2004; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). to a subset of mammalian proteins scattered in the cytosol was found to be colocalized with dots, we mammalian proteins at a perinuclear Analysis of these proteins may provide clues about the of membrane in mammalian of APG9L2 in and autophagy to be dispensable for mammalian embryonic development, it remains to be it has a role in the of in which autophagic is to be In autophagy can be induced conditions such as nutrient it also occurs at in tissues and to the of cytoplasmic (15Shintani T. Klionsky D.J. Science. 2004; 306: 990-995Crossref PubMed Scopus (2163) Google Scholar). In transgenic mice autophagy is shown to be in several tissues of nutrient conditions (11Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1918) Google Scholar), that autophagic may be required for normal in such We found that APG9L2 can function in autophagosome formation at the cellular but its in in placenta and pituitary remain to be In the of placenta from mice by autophagosome formation was and N. This the that the of APG9L2 expression in placenta and pituitary are involved in autophagic but some other membrane events. Yeast Atg9p is shown to be essential for both pathway and autophagy T. J. M. C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar), is for the pathway in eukaryotic species other (5Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3186) Google Scholar). APG9L2 may be involved in an mammalian in the tissues such as placenta and pituitary gland. of autophagy and APG9L2 in the of normal and disease conditions would into roles at the We Mizushima for on the transgenic Ohsumi for the yeast for in and for