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N Mizushima

Publications and source records attributed to N Mizushima.

At least 19 recordsLinked to original sources
PubMed2005

The pleiotropic role of autophagy: from protein metabolism to bactericide.

Autophagy is in principle a nonselective, bulk degradation system within cells, with a contribution to intracellular protein degradation estimated to be as large as that of the ubiquitin--proteasome system. The primary roles of autophagy are baseline turnover of intracellular proteins and organelles, production of amino acids in nutrient emergency, and regression of retired tissues. These functions guarantee rejuvenation and adaptation to adverse conditions, and even underlie dynamic processes such as development/metamorphosis. In addition, several other roles for autophagy have recently been discovered, such as presentation of endogenous antigens and degradation of invasive bacteria. This review will discuss the biological significance of autophagy from yeast to higher eukaryotes.

Animals↗
PubMed2001

The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation.

Macroautophagy is a bulk degradation process induced by starvation in eukaryotic cells. In yeast, 15 Apg proteins coordinate the formation of autophagosomes. Several key reactions performed by these proteins have been described, but a comprehensive understanding of the overall network is still lacking. Based on Apg protein localization, we have identified a novel structure that functions in autophagosome formation. This pre-autophagosomal structure, containing at least five Apg proteins, i.e. Apg1p, Apg2p, Apg5p, Aut7p/Apg8p and Apg16p, is localized in the vicinity of the vacuole. Analysis of apg mutants revealed that the formation of both a phosphatidylethanolamine-conjugated Aut7p and an Apg12p- Apg5p conjugate is essential for the localization of Aut7p to the pre-autophagosomal structure. Vps30p/Apg6p and Apg14p, components of an autophagy- specific phosphatidylinositol 3-kinase complex, Apg9p and Apg16p are all required for the localization of Apg5p and Aut7p to the structure. The Apg1p protein kinase complex functions in the late stage of autophagosome formation. Here, we present the classification of Apg proteins into three groups that reflect each step of autophagosome formation.

Autophagy↗
PubMed2001

Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.

Animals↗
PubMed2000

A ubiquitin-like system mediates protein lipidation.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.

Amino Acid Sequence↗
PubMed2000

LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.

Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.

Amino Acid Sequence↗
PubMed2000

The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway.

Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.

Amino Acid Sequence↗
PubMed2000

A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes.

Protein conjugation, such as ubiquitination, is the process by which the C-terminal glycine of a small modifier protein is covalently attached to target protein(s) through sequential reactions with an activating enzyme and conjugating enzymes. Here we report on a novel protein conjugation system in yeast. A newly identified ubiquitin related modifier, Urm1 is a 99-amino acid protein terminated with glycine-glycine. Urm1 is conjugated to target proteins, which requires the C-terminal glycine of Urm1. At the first step of this reaction, Urm1 forms a thioester with a novel E1-like protein, Uba4. Deltaurm1 and Deltauba4 cells showed a temperature-sensitive growth phenotype. Urm1 and Uba4 show similarity to prokaryotic proteins essential for molybdopterin and thiamin biosynthesis, although the Urm1 system is not involved in these pathways. This is the fifth conjugation system in yeast, following ubiquitin, Smt3, Rub1, and Apg12, but it is unique in respect to relation to prokaryotic enzyme systems. This fact may provide an important clue regarding evolution of protein conjugation systems in eukaryotic cells.

Amino Acid Sequence↗
PubMed2000

The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells.

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1-localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2000

Apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways.

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5(ts). Biochemical analysis of API import in the apg5(ts) strain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in the apg5(ts) mutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.

Aminopeptidases↗
PubMed1999

Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast.

Autophagy is a cellular process for bulk degradation of cytoplasmic components. The attachment of Apg12p, a modifier with no significant similarity to ubiquitin, to Apg5p is crucial for autophagy in yeast. This reaction proceeds in a ubiquitination-like manner, and requires Apg7p and Apg10p. Apg7p exhibits a considerable similarity to ubiquitin-activating enzyme (E1) and is found to activate Apg12p with ATP hydrolysis. Apg10p, on the other hand, shows no significant similarity to other proteins whose functions are known. Here, we show that after activation by Apg7p, Apg12p is transferred to the Cys-133 residue of Apg10p to form an Apg12p-Apg10p thioester. Cells expressing Apg10p(C133S) do not generate the Apg12p-Apg5p conjugate, which leads to defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These findings indicate that Apg10p is a new type of protein-conjugating enzyme that functions in the Apg12p-Apg5p conjugation pathway.

Amino Acid Sequence↗
PubMed1999

Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway.

Autophagy is an intracellular bulk degradation system that is ubiquitous for eukaryotic cells. In this process, cytoplasmic components are enclosed in autophagosomes and delivered to lysosomes/vacuoles. We recently found that a protein conjugation system, in which Apg12p is covalently attached to Apg5p, is indispensable for autophagy in yeast. Here, we describe a novel coiled-coil protein, Apg16p, essential for autophagy. Apg16p interacts with Apg12p-conjugated Apg5p and less preferentially with unconjugated Apg5p. Moreover, the coiled-coil domain of Apg16p mediates self-multimerization that leads to cross-linking of Apg5p molecules and formation of a stable protein complex. Apg16p is not essential for the Apg12p-Apg5p conjugation reaction. These results suggest that the Apg12p-Apg5p conjugate requires Apg16p to accomplish its role in the autophagy pathway, and Apg16p is a key molecule as a linker to form the Apg12p-Apg5p-Apg16p multimer.

Amino Acid Sequence↗
PubMed1999

Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy.

In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.

Adenosine Triphosphate↗
PubMed1999

Lipid microsphere preparation of a lipophilic ceramide derivative suppresses colony formation in a murine experimental pulmonary metastasis model.

Ceramide is a well-known regulator of apoptosis and cell growth. In this study, we synthesized lipophilic ceramide derivatives to incorporate into lipid microspheres (LM) and their activity was evaluated in vivo. Cera 03, a lipophilic ceramide derivative synthesized from membrane-permeable C2-ceramide, caused potent growth inhibition and DNA fragmentation of Meth A-T tumor cells in vitro. Its potency was similar to that of C2-ceramide. Both compounds increased the proportion of apoptotic cells. Cera 02, the diacetylated form of natural ceramide (Cer), also suppressed in vitro cell growth with a similar or higher potency to that of Cer, but both were far less potent than C2-ceramide and Cera 03. LM containing Cera 03 (Lipo-Cera 03) could not totally prevent metastatic incidence of Meth A-T cells, but reduced pulmonary metastatic nodules in number. Intravenous injection of Lipo-Cera 03 (1 mg/kg of Cera 03) produced about 35% inhibition, while Lipo-Cera 02 had no significant effect. In conclusion, Lipo-Cera 03 may have potential as an antimetastatic drug and may also be a useful tool for researching the role of ceramides in vivo.

Animals↗
PubMed1998

A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essential for autophagy.

Autophagy is an intracellular process for bulk degradation of cytoplasmic components. We recently found a protein conjugation system essential for autophagy in the yeast, Saccharomyces cerevisiae. The C-terminal glycine of a novel modifier protein, Apg12p, is conjugated to a lysine residue of Apg5p via an isopeptide bond. This conjugation reaction is mediated by Apg7p, a ubiquitin activating enzyme (E1)-like enzyme, and Apg10p, suggesting that it is a ubiquitination-like system (Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M. , and Ohsumi, Y. (1998) Nature 395, 395-398). Although autophagy is a ubiquitous process in eukaryotic cells, no molecule involved in autophagy has yet been identified in higher eukaryotes. We reasoned that this conjugation system could be conserved. Here we report cloning and characterization of the human homologue of Apg12 (hApg12). It is a 140-amino acid protein and possesses 27% identity and 48% similarity with the yeast Apg12p, but no apparent homology to ubiquitin. Northern blot analysis showed that its expression was ubiquitous in human tissues. We found that it was covalently attached to another protein. This target protein was identified to be the human Apg5 homologue (hApg5). Mutagenic analyses suggested that this conjugation was formed via an isopeptide bond between the C-terminal glycine of hApg12 and Lys-130 of hApg5. These findings indicate that the Apg12 system is well conserved and may function in autophagy also in human cells.

Adult↗
PubMed1998

A protein conjugation system essential for autophagy.

Autophagy is a process for the bulk degradation of proteins, in which cytoplasmic components of the cell are enclosed by double-membrane structures known as autophagosomes for delivery to lysosomes or vacuoles for degradation. This process is crucial for survival during starvation and cell differentiation. No molecules have been identified that are involved in autophagy in higher eukaryotes. We have isolated 14 autophagy-defective (apg) mutants of the yeast Saccharomyces cerevisiae and examined the autophagic process at the molecular level. We show here that a unique covalent-modification system is essential for autophagy to occur. The carboxy-terminal glycine residue of Apg12, a 186-amino-acid protein, is conjugated to a lysine at residue 149 of Apg5, a 294-amino-acid protein. Of the apg mutants, we found that apg7 and apg10 were unable to form an Apg5/Apg12 conjugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-E1-like enzyme. This conjugation can be reconstituted in vitro and depends on ATP. To our knowledge, this is the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system. Furthermore, Apg5 and Apg12 have mammalian homologues, suggesting that this new modification system is conserved from yeast to mammalian cells.

Amino Acid Sequence↗
PubMed1998

Ceramide, a mediator of interleukin 1, tumour necrosis factor alpha, as well as Fas receptor signalling, induces apoptosis of rheumatoid arthritis synovial cells.

OBJECTIVES: To examine the effects of ceramide, which is a lipid second messenger of cell surface receptors, including tumour necrosis factor alpha (TNF alpha), interleukin 1 (IL1), and Fas receptors, on rheumatoid arthritis (RA) synovial cells. METHODS: Synovial cells from RA patients and normal skin fibroblasts were cultured with cell permeable ceramide (C2-ceramide). Apoptosis was assessed by microscopic observation of morphological changes, nuclear staining, and DNA electrophoresis. DNA synthesis was examined by thymidine incorporation. RESULTS: C2-ceramide induced reversible morphological changes of synovial cells such as cell rounding within four hours. Subsequently, irreversible nuclear changes characteristic to apoptosis were observed at 48 hours. DNA synthesis was not promoted. The addition of ceramide exerted similar effects on cultured dermal fibroblasts. CONCLUSION: Ceramide induced apoptosis in RA synovial cells. Ceramide could be a second messenger specific for apoptosis of RA synovial cells.

Apoptosis↗