[Show abstract][Hide abstract] ABSTRACT: The Saccharomyces cerevisiae autophagy-initiation complex, Atg1 kinase complex, consists of Atg1, Atg13, Atg17, Atg29, and Atg31, while the corresponding complex in most other eukaryotes, including mammals, is composed of ULK1 (or ULK2), Atg13, FIP200 (also known as RB1CC1), and Atg101. ULKs are homologs of Atg1, and FIP200 is partially homologous to Atg17. However, the sequence of Atg101 is not similar to that of Atg29 or Atg31. Although Atg101 is essential for autophagy and widely conserved in eukaryotes, its precise function and structure have remained largely unknown. Now, structural and cell biological analysis of Atg101 together with its binding partner Atg13 reveal that Atg101 is required for stabilization of "uncapped" Atg13 in most eukaryotes and also for recruitment of downstream Atg proteins through the newly identified WF motif. By contrast, S. cerevisiae has stable "capped" Atg13, which does not require Atg101 for its stabilization. Possible roles for other binding partners such as Atg29, Atg31, and Atg28 in different organisms are also discussed.
Preview · Article · Jan 2016 · Cell Structure and Function
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a major intracellular degradation system by which cytoplasmic components are enclosed by autophagosomes and delivered
to lysosomes. Formation of the autophagosome requires a set of autophagy-related (Atg) proteins. Among them, the ULK1 complex,
which is composed of ULK1 (or ULK2), FIP200, Atg13, and Atg101, acts at an initial step. Previous studies showed that ULK1
and FIP200 also function in pathways other than autophagy. However, whether Atg13 and Atg101 act similarly to ULK1 and FIP200
remains unknown. In the present study, we generated Atg13 knockout mice. Like FIP200-deficient mice, Atg13-deficient mice die in utero, which is distinct from most other types of Atg-deficient mice. Atg13-deficient embryos show growth retardation and myocardial growth defects. In cultured fibroblasts, Atg13 deficiency blocks
autophagosome formation at an upstream step. In addition, sensitivity to TNF-α-induced apoptosis is enhanced by deletion of
Atg13 or FIP200, but not by other Atg proteins, as well as by simultaneous deletion of ULK1 and ULK2. These results suggest
that Atg13 has both autophagic and non-autophagic functions, and that the latter is essential for cardiac development and
likely shared with FIP200 but not with ULK1/2.
No preview · Article · Dec 2015 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: The Atg1/ULK complex functions as the most upstream factor among Atg proteins to initiate autophagy. ATG101 is a constitutive component of the Atg1/ULK complex in most eukaryotes except for budding yeast, and plays an essential role in autophagy; however, the structure and functions of ATG101 were largely unknown. Recently, we determined the crystal structure of fission yeast Atg101 in complex with the closed HORMA domain of Atg13, revealing that Atg101 is also a HORMA protein with an open conformation. These 2 HORMA proteins play essential roles in autophagy initiation through recruiting downstream factors to the autophagosome formation site.
[Show abstract][Hide abstract] ABSTRACT: Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1 and ATG101 has been identified as central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases like MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.
[Show abstract][Hide abstract] ABSTRACT: Atg101 is an essential component of the autophagy-initiating ULK complex in higher eukaryotes, but it is absent from the functionally equivalent Atg1 complex in budding yeast. Here, we report the crystal structure of the fission yeast Atg101-Atg13 complex. Atg101 has a Hop1, Rev7 and Mad2 (HORMA) architecture similar to that of Atg13. Mad2 HORMA has two distinct conformations (O-Mad2 and C-Mad2), and, intriguingly, Atg101 resembles O-Mad2 rather than the C-Mad2-like Atg13. Atg13 HORMA from higher eukaryotes possesses an inherently unstable fold, which is stabilized by Atg101 via interactions analogous to those between O-Mad2 and C-Mad2. Mutational studies revealed that Atg101 is responsible for recruiting downstream factors to the autophagosome-formation site in mammals via a newly identified WF finger. These data define the molecular functions of Atg101, providing a basis for elucidating the molecular mechanisms of mammalian autophagy initiation by the ULK complex.
No preview · Article · Jun 2015 · Nature Structural & Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: WDR45/WIPI4, encoding a WD40 repeat-containing PtdIns(3)P binding protein, is essential for the basal autophagy pathway. Mutations in WDR45 cause the neurodegenerative disease beta-propeller protein-associated neurodegeneration (BPAN), a subtype of NBIA. We generated CNS-specific Wdr45 knockout mice, which exhibit poor motor coordination, greatly impaired learning and memory, and extensive axon swelling with numerous axon spheroids. Autophagic flux is defective and SQSTM1 (sequestosome-1)/p62 and ubiquitin-positive protein aggregates accumulate in neurons and swollen axons. Nes-Wdr45(fl/Y) mice recapitulate some hallmarks of BPAN, including cognitive impairment and defective axonal homeostasis, providing a model for revealing the disease pathogenesis of BPAN and also for investigating the possible role of autophagy in axon maintenance.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is an intracellular catabolic system that degrades cytoplasmic proteins and organelles. Damaged mitochondria can be degraded by a selective type of autophagy, which is termed mitophagy. PINK1-Parkin-dependent mitophagy has been extensively studied in the mammalian system. PINK1 accumulates on damaged mitochondria to recruit Parkin, which subsequently ubiquitinates a broad range of outer mitochondrial membrane proteins. Ubiquitinated mitochondria associate with the autophagosome formation site, and are selectively incorporated into autophagosomes. During this process, damaged mitochondria first associate with the autophagosome formation site together with upstream autophagy factors, then are efficiently incorporated into autophagosomes through binding with autophagosome adaptors. This "two-step model" may be applied to other selective types of autophagy. This article is part of a Special Issue entitled: Mitophagy.
No preview · Article · Jan 2015 · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
[Show abstract][Hide abstract] ABSTRACT: Autophagy is an intracellular degradation system that is mediated by orchestrated functions of membranes and proteins. A genetic screen in Caenorhabditis elegans revealed that O-linked N-acetylglucosamine modification of the SNARE protein SNAP-29 negatively regulates SNARE-dependent fusion between autophagosomes and lysosomes. This regulatory mechanism is conserved in mammals.
No preview · Article · Dec 2014 · Nature Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial
homeostasis, including mitochondrial DNA (mtDNA) distribution. Cardiac myocytes have a specialized cytoplasmic structure where
large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial
dynamics also plays an important role in the formation and maintenance of cardiomyocytes. Here, we precisely analyzed the
role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in the developing neonatal heart, and muscle-specific Drp1 knockout
(Drp1-KO) mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes
resulted in severe mtDNA nucleoid clustering and led to mosaic deficiency of mitochondrial respiration. The functional and
structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus,
the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting
homogeneous distribution of active mitochondria throughout the cardiomyocytes.
No preview · Article · Oct 2014 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Autophagy maintains cell and tissue homeostasis through catabolic degradation. To better delineate the in vivo function for autophagy in adaptive responses to tissue injury, we examined the impact of compromised autophagy in mouse submandibular glands (SMGs) subjected to main excretory duct ligation. Blocking outflow from exocrine glands causes glandular atrophy by increased ductal pressure. Atg5(f/-);Aqp5-Cre mice with salivary acinar-specific knockout (KO) of autophagy essential gene Atg5 were generated. While duct ligation induced autophagy and the expression of inflammatory mediators, SMGs in Atg5(f/-);Aqp5-Cre mice, before ligation, already expressed higher levels of proinflammatory cytokine and Cdkn1a/p21 messages. Extended ligation period resulted in the caspase-3 activation and acinar cell death, which was delayed by Atg5 knockout. Moreover, expression of a set of senescence-associated secretory phenotype (SASP) factors was elevated in the post-ligated glands. Dysregulation of cell-cycle inhibitor CDKN1A/p21 and activation of senescence-associated β-galactosidase were detected in the stressed SMG duct cells. These senescence markers peaked at day 3 after ligation and partially resolved by day 7 in post-ligated SMGs of wild-type (WT) mice, but not in KO mice. The role of autophagy-related 5 (ATG5)-dependent autophagy in regulating the tempo, duration and magnitude of cellular stress responses in vivo was corroborated by in vitro studies using MEFs lacking ATG5 or autophagy-related 7 (ATG7) and autophagy inhibitors. Collectively, our results highlight the role of ATG5 in the dynamic regulation of ligation-induced cellular senescence and apoptosis, and suggest the involvement of autophagy resolution in salivary repair.
Full-text · Article · Oct 2014 · Cell Death & Disease
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation [1 and 2]. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1 [3 and 4], as well as analyzing a human patient harboring a point mutation in Drp1 , showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically [6 and 7], and their organelle aggregation is related to germ cell formation and epigenetic regulation [8, 9 and 10]. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an age-dependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca2+ signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement.
[Show abstract][Hide abstract] ABSTRACT: Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as /`accidental cell death/' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. /`Regulated cell death/' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects i
Full-text · Article · Sep 2014 · Cell death and differentiation
[Show abstract][Hide abstract] ABSTRACT: The ATG genes are highly conserved in eukaryotes including yeasts, plants, and mammals. However, these genes appear to be only partially present in most protists. Recent studies demonstrated that, in the apicomplexan parasites Plasmodium (malaria parasites) and Toxoplasma, ATG8 localizes to the apicoplast, a unique nonphotosynthetic plastid with 4 limiting membranes. In contrast to this established localization, it remains unclear whether these parasites can induce canonical macroautophagy and if ATG8 localizes to autophagosomes. Furthermore, the molecular function of ATG8 in its novel workplace, the apicoplast, is totally unknown. Here, we review recent studies on ATG8 in Plasmodium and Toxoplasma, summarize both consensus and controversial findings, and discuss its potential role in these parasites.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is mediated by a unique organelle, the autophagosome. Autophagosome formation involves a number of autophagy-related (ATG) proteins and complicated membrane dynamics. Although the hierarchical relationships of ATG proteins have been investigated, how individual ATG proteins or their complexes contribute to the organization of the autophagic membrane remains largely unknown. Here, systematic ultrastructural analysis of mouse embryonic fibroblasts and HeLa cells deficient in various ATG proteins revealed that the emergence of the isolation membrane (phagophore) requires FIP200/RB1CC1, ATG9A, and PtdIns 3-kinase activity. By contrast, small premature isolation membrane- and autophagosome-like structures were generated in cells lacking VMP1 and ATG2A/B, respectively. The isolation membranes could elongate in cells lacking ATG5, but these did not mature into autophagosomes. We also found that ferritin clusters accumulated at the autophagosome formation site together with p62/SQSTM1 in autophagy-deficient cells. These results reveal the specific functions of these representative ATG proteins in autophagic membrane organization and ATG-independent recruitment of ferritin to the autophagosome formation site.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3 (GFP-LC3 MEFs). One of the inhibitors we identified, 2, 5-pyridinedicarboxamide, N2, N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron-microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mTOR activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a dynamically regulated intracellular degradation system that is important for cellular processes such as amino acid production during starvation and intracellular quality control. Previously, we reported that autophagy is suppressed in oocytes, but is rapidly up-regulated after fertilization. During this period, autophagy is thought to be important for the generation of amino acids from the bulk degradation of maternal proteins that have accumulated during oogenesis. However, the mechanism of autophagy induction after fertilization is presently unknown. In most cell types, autophagy is negatively controlled by mammalian target of rapamycin complex 1 (mTORC1), which is typically regulated by amino acids and insulin or related growth factors. In this study, we determined the role of mTORC1 in fertilization-induced autophagy. On the basis of the phosphorylation status of mTORC1 substrates, we found that mTORC1 activity was relatively high in metaphase II (MII) oocytes, but was rapidly decreased within 3 h of fertilization. However, chemical inhibition of mTORC1 by Torin1 or PP242 in MII oocytes or fertilized embryos did not induce autophagy. In addition, activation of mTORC1 by cycloheximide did not inhibit fertilization-induced autophagy in fertilized embryos. By contrast, the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 effectively suppressed autophagy in these embryos. These data suggest that, even though autophagy induction and post-fertilization mTORC1 activity are inversely correlated with each other, as observed in other cell types, mTORC1 suppression is neither essential nor sufficient for fertilization-induced autophagy, highlighting a unique feature of the regulation mechanism of autophagy-mediated intracellular turnover in early embryos.
Preview · Article · May 2014 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.
Preview · Article · Mar 2014 · Biochemical and Biophysical Research Communications