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Dissection And Reconstitution Of Autophagic Membrane Fusion
Author(s) -
Zhong Qing,
Diao Jiaojie,
Liu Rong,
Rong Yueguang
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.502.1
Subject(s) - microbiology and biotechnology , snare complex , lipid bilayer fusion , autophagy , autophagosome , lysosome , fusion protein , tethering , chemistry , signal transducing adaptor protein , biogenesis , biology , biochemistry , membrane , signal transduction , recombinant dna , apoptosis , gene , enzyme
Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. We recently reported that ATG14/Barkor/ATG14L, an essential autophagy‐specific regulator of the class III phosphatidylinositol 3‐kinase complex, promotes membrane tethering of protein‐free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)‐SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)‐SNARE VAMP8. ATG14 binds to the SNARE core domain of STX17 through its coiled‐coil domain, and stabilizes the STX17‐SNAP29 binary t‐SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion‐enhancing activities of ATG14 require its homo‐oligomerization by cysteine repeats. In ATG14 homo‐oligomerization‐defective cells, autophagosomes still efficiently form but their fusion with lysosomes is blocked. Recombinant ATG14 homo‐oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE‐mediated fusion in vitro. Our data suggest an autophagy‐specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17‐SNAP29 binary t‐SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome‐lysosome fusion. We are currently investigating protein factors and likely protein modifications required for full reconstitution of autophagosomal fusion with lysosomes. Support or Funding Information The work was supported by grants to Q. Z. from the Welch Foundation (I‐1864), CPRIT (RP140320), and NIH R01 (GM116908).