Abstract
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 the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed 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.
Highlights
IntroductionPivotal 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 the lysosome/late endosome for degradation
Pivotal to the maintenance of cellular homeostasis, macroautophagy is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation
Recent studies on C. elegans EPG-5 by the Zhang group led to the proposal that this large-sized protein serves as an autophagy tethering factor, as it possesses two features found in tethering factors of other membrane trafficking pathways: (1) the ability to bind the transport vesicle and the target organelle
Summary
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 the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed 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. The discovery of the ATG (autophagy related) genes by yeast genetic screening and the identification of the core autophagy machinery composed of 18 mostly conserved Atg proteins generated a framework for investigating the molecular mechanism of this multistep degradation pathway. Subsequent studies in C. elegans and human cell lines showed that EPG5 is recruited to the lysosome/late endosome by the small GTPase RAB7, and EPG5 has the ability to bind the autophagosome surface protein and ATG8 homolog human LC3B or C. elegans LGG-1 via two LC3-interacting region (LIR) motifs composed of a conserved sequence [(W/F/Y)-X1-X2(I/L/V)]20. Together with the finding that C. elegans EPG-5 is capable of stabilizing and facilitating assembly of the STX17–SNAP29–VAMP8 trans-SNARE complex in vitro, these new data led to the model that EPG5 functions as an autophagy tethering factor that mediates initial interaction between the autophagosome and the lysosome[20]
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