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Lysosome Tethering from Structural and Biochemical Characterization of Human Autophagy Factor EPG5

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Lysosome Tethering from Structural and Biochemical Characterization of Human Autophagy Factor EPG5 ARTICLE https://doi.org/10.1038/s42003-021-01830-x OPEN Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5 Sung-Eun Nam1,3, Yiu Wing Sunny Cheung 1,3, Thanh Ngoc Nguyen 2, Michael Gong1, Samuel Chan1, ✉ Michael Lazarou 2 & Calvin K. Yip 1 Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to 1234567890():,; the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein pro- posed to serve as a tethering factor to enforce autophagosome–lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended “shepherd’s staff” architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome–lysosome fusion. The hEPG5–GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5’s overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5. 1 Life Sciences Institute, Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, BC, Canada. 2 Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, Australia. 3These authors contributed equally: ✉ Sung-Eun Nam, Yiu Wing Sunny Cheung. email: [email protected] COMMUNICATIONS BIOLOGY | (2021) 4:291 | https://doi.org/10.1038/s42003-021-01830-x | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-01830-x acroautophagy (also known as autophagy) is the main lines showed that EPG5 is recruited to the lysosome/late endo- Mpathway for degrading long-lived cytoplasmic macro- some by the small GTPase RAB7, and EPG5 has the ability to molecules and full-sized organelles and represents a key bind the autophagosome surface protein and ATG8 homolog component of the cellular homeostatic program. Under favorable human LC3B or C. elegans LGG-1 via two LC3-interacting region growth conditions, basal autophagy serves as a quality control (LIR) motifs composed of a conserved sequence [(W/F/Y)-X1-X2- mechanism to selectively remove misfolded/aggregated proteins (I/L/V)]20. Together with the finding that C. elegans EPG-5 is and dysfunctional organelles in the cytoplasm1,2. When cells capable of stabilizing and facilitating assembly of the encounter stress conditions, such as starvation, autophagy is STX17–SNAP29–VAMP8 trans-SNARE complex in vitro, these upregulated to promote nonselective bulk degradation to generate new data led to the model that EPG5 functions as an autophagy basic building blocks to power essential metabolic reactions and tethering factor that mediates initial interaction between the to generate energy3–5. Because of autophagy’s important roles in autophagosome and the lysosome20. guarding normal cellular physiology, dysregulation of this At around the time when EPG5’s role in autophagy was degradation pathway is linked to many human pathologies ran- uncovered, clinical genetics analysis revealed that recessive ging from neurodegeneration and cancer to infectious diseases6,7. mutations of the gene encoding human EPG5 (hEPG5) cause Vici An improved understanding of autophagy at the molecular level syndrome, a rare but severe multisystem disorder characterized will generate insights into the basis of different human diseases by agenesis of the corpus callosum, cataracts, cardiomyopathy, and may reveal additional avenues for therapeutic intervention. hypopigmentation, and combined immunodeficiency22–24. Autophagy degradation begins with the formation of a mem- Approximately 100 cases of Vici syndrome have been reported to brane precursor known as the phagophore. The phagophore date with a median survival time of 24 months22–24. Analyses of expands in size, sequesters cytoplasmic materials, and self-seals to primary cells isolated from patients showed an accumulation of form a double-membrane transport vesicle called the autopha- autophagosomes attributed to deficiency in autophagosome– gosome. The cargo-laden autophagosome then gets transported lysosome fusion24,25. Interestingly, epg5−/− knockout mice to and fuses with the lysosome or the late endosome, where the exhibit neurodegenerative features resembling human amyo- content of the autophagosome is ultimately digested by hydrolytic trophic lateral sclerosis26. Although many Vici syndrome muta- enzymes inside the lysosome3–5,8. The discovery of the ATG tions have been mapped, the effects of these mutations on EPG5’s (autophagy related) genes by yeast genetic screening and the structure and function are not known. identification of the core autophagy machinery composed of 18 By developing a method to produce recombinant full-length mostly conserved Atg proteins generated a framework for hEPG5, we were able to comprehensively characterize the struc- investigating the molecular mechanism of this multistep degra- tural and biochemical properties of this large-sized putative dation pathway. Subsequent characterization of the core autop- autophagy tethering factor. We found that hEPG5 adopts an hagy machinery consisting of five key functional groups (Atg1 extended architecture reminiscent to tethering factors found in kinase complex, autophagy-specific phosphatidylinositol 3- other membrane trafficking pathways. We also found that kinase/PI3K complex, Atg8 conjugation system, Atg12 conjuga- hEPG5 shows preferential binding to the GABARAP subfamily of tion system, Atg9 and Atg2–Atg18 complex) yielded mechanistic ATG8 proteins, and this interaction involves a complex interplay insights into autophagy initiation and autophagosome biogenesis, between the two LIR motifs exhibiting differential binding affi- primarily in the yeast model system3,9,10. However, the nities. We further showed that hEPG5–GABARAP interaction is mechanisms underlying the later steps of autophagy, including required for hEPG5 recruitment to mitochondria during PINK1/ how autophagosome engages and ultimately fuses with the lyso- Parkin-mediated mitophagy. Lastly, the common recurrent Vici some remain less well understood. syndrome mutation Q336R did not affect the overall architecture, Recent studies in high eukaryotes have begun to unravel these stability, and GABARAP-binding ability of hEPG5. mysteries. The identification of syntaxin 17 (STX17) and YKT6 as the autophagosomal “SNARE” that bind lysosomal VAMP8– SNAP29 and STX7–SNAP29, respectively, to form trans-SNARE Results complexes offered insights into the autophagosome–lysosome hEPG5 adopts an extended overall architecture. With 2579 membrane fusion process11,12. Systematic gene deletion studies of amino acid residues and an overall molecular mass of ~290 kDa, the six homologs of human ATG8 (LC3A, LC3B, LC3C, hEPG5 is one of the largest regulators in the autophagy pathway GABARAP, GABARAPL1, and GABARAPL2) revealed that the identified to date. Due in part to technical challenges associated three members of the GABARAP subfamily play critical roles to with purification of this large-sized protein, nothing is currently autophagosome–lysosome fusion13. Furthermore, the discovery known about the structural properties of hEPG5. To overcome of new non-ATG autophagy regulators, including the conserved this barrier, we developed a baculovirus-insect cell-based system multi-subunit HOPS complex, and the metazoan-specific proteins to overexpress the recombinant full-length hEPG5 and an anti- TECPR1, PLEKHM1, BRUCE, GRASP55, and EPG5 generated a FLAG affinity chromatography coupled with glycerol density growing list of additional proteins/protein complexes that parti- ultracentrifugation approach to purify the recombinant protein. cipate in the terminal stage of autophagy11,14–20. However, the This procedure enabled us to obtain highly purified precise physiological functions of these newly identified autop- hEPG5 suitable for biochemical and structural characterization hagy factors, and exactly how they coordinate with one another (Fig. 1a). Analytical gel filtration chromatography of purified and the GABARAP proteins to mediate autophagosome– hEPG5 showed that it elutes at a volume corresponding to a lysosome fusion are not fully understood. predicted molecular weight higher than its calculated mass Originally discovered in the Caenorhabditis elegans genetic (Fig. 1b). This suggests that hEPG5 either exists as an obligate screen for metazoan autophagy genes, EPG5 (ectopic P-granules oligomer or adopts a non-globular overall shape. We next autophagy protein 5) is a large-sized (~292 kDa) protein pro- examined hEPG5 by negative stain single-particle electron posed to regulate fusion specificity between autophagosomes and microscopy (EM). Raw images not only revealed highly elongated lysosomes. Notably, epg-5 deficiency in C. elegans causes non- particles with a distinct curvature at one end,
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