An Atg4B Mutant Hampers the Lipidation of LC3 Paralogues and Causes Defects in Autophagosome Closure

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Molecular biology of the cell (Impact Factor: 4.47). 10/2008; 19(11):4651-9. DOI: 10.1091/mbc.E08-03-0312
Source: PubMed


In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.

Download full-text


Available from: Takeshi Noda
  • Source
    • "The bulk protein degradation assay was performed according to the method described by Fujita et al. [21] with some modifications. Briefly, cells (1 × 106 cells) were seeded in 35 mm culture dishes and incubated overnight. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE (-/-) ) mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE (-/-) macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE (-/-) macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR), Akt, and extracellular signal-related kinase (ERK). Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE (-/-) macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE (-/-) cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE (-/-) macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE (-/-) macrophages showed increased reactive oxygen species (ROS) production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.
    Full-text · Article · Dec 2013 · PLoS ONE
  • Source
    • "The constructed autophagy-deficient cell line, HTR8-ATG4BC74A mutant cells, and the control vector-infected cell line, HTR8-mStrawberry cells, were also used. The procedures for constructing the vectors were reported previously [21]. The expression of mStrawberry was confirmed by fluorescence microscopy. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Extravillous trophoblasts (EVTs) characterize the invasion of the maternal decidua under low oxygen and poor nutrition at the early feto-maternal interface to establish a successful pregnancy. We previously reported that autophagy in EVTs was activated under 2% O2 in vitro, and autophagy activation was also observed in EVTs at the early feto-maternal interface in vivo. Here, we show that autophagy is an energy source for the invasion of EVTs. Cobalt chloride (CoCl2), which induces hypoxia inducible factor 1α (HIF1α) overexpression, activated autophagy in HTR8/SVneo cells, an EVT cell line. The number of invading HTR8-ATG4BC74A cells, an autophagy-deficient EVT cell line, was markedly reduced by 81 percent with the CoCl2 treatment through the suppression of MMP9 level, although CoCl2 did not affect the cellular invasion of HTR8-mStrawberry cells, a control cell line. HTR8-ATG4BC74A cells treated with CoCl2 showed a decrease in cellular adenosine triphosphate (ATP) levels and a compensatory increase in the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2RX7), which is stimulated with ATP, whereas HTR8-mStrawberry cells maintained cellular ATP levels and did not affect P2RX7 expression. Furthermore, the decreased invasiveness of HTR8-ATG4BC74A cells treated with CoCl2 was neutralized by ATP supplementation to the level of HTR8-ATG4BC74A cells treated without CoCl2. These results suggest that autophagy plays a role in maintaining homeostasis by countervailing HIF1α-mediated cellular energy consumption in EVTs.
    Preview · Article · Oct 2013 · PLoS ONE
  • Source
    • "As shown in Fig. 7C, GFP-Atg4B alone indeed increased LC3-I level. However, this increase in LC3-I is likely due to cleavage of LC3-II by the excess of GFP-Atg4B [22], which is also supported by little amount of LC3-II in the cells with over-expression of GFP-Atg4B (lane 2 and lane 5). However, GFP-Atg4BC74A mutant indeed resulted in increase of LC3-I levels in GFP-Atg4BC74A alone and GFP-Atg4BC74A/mRFP-p62 expression settings (Fig. 7C, lane 3 and lane 6). "
    [Show abstract] [Hide abstract]
    ABSTRACT: p62 is constitutively degraded by autophagy via its interaction with LC3. However, the interaction of p62 with LC3 species in the context of the LC3 lipidation process is not specified. Further, the p62-mediated protein aggregation's effect on autophagy is unclear. We systemically analyzed the interactions of p62 with all known Atg proteins involved in LC3 lipidation. We find that p62 does not interact with LC3 at the stages when it is being processed by Atg4B or when it is complexed or conjugated with Atg3. p62 does interact with LC3-I and LC3-I:Atg7 complex and is preferentially recruited by LC3-II species under autophagic stimulation. Given that Atg4B, Atg3 and LC3-Atg3 are indispensable for LC3-II conversion, our study reveals a protective mechanism for Atg4B, Atg3 and LC3-Atg3 conjugate from being inappropriately sequestered into p62 aggregates. Our findings imply that p62 could potentially impair autophagy by negatively affecting LC3 lipidation and contribute to the development of protein aggregate diseases.
    Preview · Article · Sep 2013 · PLoS ONE
Show more