The WD40 Repeat PtdIns(3)P-Binding Protein EPG-6 Regulates Progression of Omegasomes to Autophagosomes

National Institute of Biological Sciences, Beijing 102206, People's Republic of China.
Developmental Cell (Impact Factor: 9.71). 08/2011; 21(2):343-57. DOI: 10.1016/j.devcel.2011.06.024
Source: PubMed


PtdIns(3)P plays critical roles in the autophagy pathway. However, little is known about how PtdIns(3)P effectors act with autophagy proteins in autophagosome formation. Here we identified an essential autophagy gene in C. elegans, epg-6, which encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function causes accumulation of enlarged early autophagic structures. Another WD40 repeat PtdIns(3)P effector, ATG-18, plays a distinct role in autophagosome formation. We also established the hierarchical relationship of autophagy genes in degradation of protein aggregates and revealed that the UNC-51/Atg1 complex, EPG-8/Atg14, and binding of lipidated LGG-1 to protein aggregates are required for omegasome formation. Our study demonstrates that autophagic PtdIns(3)P effectors play distinct roles in autophagosome formation and also provides a framework for understanding the concerted action of autophagy genes in protein aggregate degradation.

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Available from: Attila L Kovacs, Oct 01, 2015
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    • "Although it is not possible to conclude that Atg101 is not required for Atg8a recruitment to ref(2)P aggregates solely based on RNAi experiments, this seems to be a possibility, which is also supported by data from mutant worms. Interestingly, of the core Atg genes, Atg2 has also been shown to be dispensable for Atg8a recruitment to the phagophore assembly site and protein aggregates in worm, fly, and mammalian cells [19, 24, 36, 37]. To exclude the possibility that these Atg8a dots might represent autophagosomes, we carried out electron microscopy. "
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    ABSTRACT: The large-scale turnover of intracellular material including organelles is achieved by autophagy- mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2)P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13, and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2)P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes.
    BioMed Research International 03/2014; 2014(4). DOI:10.1155/2014/470482 · 2.71 Impact Factor
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    • "In contrast , Syx17 essentially did not colocalize with the phagophore marker Atg5 (2.5%, 5/200) or GFP-dLAMP–positive late endosomes and lysosomes (6.8%, 16/236; Fig. 4, C and D). Loss of Atg2 results in accumulation of stalled phagophores that already contain Atg8 homologues in worms and mammals (Lu et al., 2011; Velikkakath et al., 2012). Accordingly, Atg8a and Syx17 did not colocalize in Atg2 mutants (2.8%, 9/322; "
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    ABSTRACT: During autophagy, phagophores capture portions of cytoplasm and form double-membrane autophagosomes to deliver cargo for lysosomal degradation. How autophagosomes gain competence to fuse with late endosomes and lysosomes is not known. In this paper, we show that Syntaxin17 is recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with ubisnap (SNAP-29) and VAMP7 in Drosophila melanogaster. Loss of these genes results in accumulation of autophagosomes and a block of autolysosomal degradation during basal, starvation-induced, and developmental autophagy. Viable Syntaxin17 mutant adults show large-scale accumulation of autophagosomes in neurons, severe locomotion defects, and premature death. These mutant phenotypes cannot be rescued by neuron-specific inhibition of caspases, suggesting that caspase activation and cell death do not play a major role in brain dysfunction. Our findings reveal the molecular mechanism underlying autophagosomal fusion events and show that lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the nervous system.
    The Journal of Cell Biology 05/2013; 201(4):531-9. DOI:10.1083/jcb.201211160 · 9.83 Impact Factor
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    • "The amino acid sequence alignment of loop 2 of the Atg18 b-propeller with that of various WIPI4 proteins from different species supports this idea, because several amino acids are well conserved except for C. elegans EPG-6 (Fig. 8C). The ATG-2- binding site of EPG-6 has been mapped to the fifth and sixth blades of the EPG-6 b-propeller, and this could explain the lack of amino acid conservation in loop 2 of EPG-6 b-propeller (Lu et al., 2011). This binding region, however, was identified by sequential deletion of the blades and therefore it cannot be excluded that this type of approach leads to a complete disruption of the b-propeller structure making the interpretation of the result difficult. "
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    ABSTRACT: Autophagy is a conserved degradative transport pathway. It is characterized by the formation of double-membrane autophagosomes at the phagophore assembly site (PAS). Atg18 is essential for autophagy but also for vacuole homeostasis and probably endosomal functions. This protein is basically a β-propeller formed by 7 WD40 repeats, which contains a conserved FRRG motif that binds phosphoinositides and promotes Atg18 recruitment to the PAS, endosomes and vacuoles. It is unknown, however, how Atg18 association with these organelles is regulated as the phosphoinositides bound by this protein are present on the surface of all of them. We have investigated Atg18 recruitment to the PAS and found that Atg18 binds Atg2 through a specific stretch of amino acids in the β-propeller on the opposite surface from the FRRG motif. As in absence of the FRRG sequence, the inability of Atg18 to interact with Atg2 impairs its association with the PAS, causing an autophagy block. Our data provide a model, where the Atg18 β-propeller provides organelle specificity by binding two determinants on the target membrane.
    Journal of Cell Science 12/2012; 126(2). DOI:10.1242/jcs.115725 · 5.43 Impact Factor
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