LC3 and GATE-16 N Termini Mediate Membrane Fusion Processes Required for Autophagosome Biogenesis

Abstract

Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion. We found that both proteins promote tethering and membrane fusion, mediated by the proteins' N-terminal α helices. We further show that short, 10 amino acid long synthetic peptides derived from the N terminus of LC3 or GATE-16 are sufficient to promote membrane fusion. Our data indicate that the fusion activity of LC3 is mediated by positively charged amino acids, whereas the activity of GATE-16 is mediated by hydrophobic interactions. Finally, we demonstrate that LC3 and GATE-16 N termini in general and specific residues needed for the fusion activity are essential for the proteins role in autophagosome biogenesis.

Membrane Tethering and Nucleotide-dependent Conformational Changes Drive Mitochondrial Genome Maintenance (Mgm1) Protein-mediated Membrane Fusion

“…To investigate the functional significance of the GTP-dependent structural transitions, we monitored potential s-Mgm1 membrane fusion activity using a well characterized NBD-rhodamine lipid mixing assay (24,29). We showed that s-Mgm1 promoted IM lipid mixing in a dose-dependent manner independently of nucleotide (Fig.…”

ATG16L1: A multifunctional susceptibility factor in Crohn disease

“…43 Upon autophagosome closure, the LC3-II on the outer surface of the autophagosome is cleaved from PE and released back into the cytosol, along with the other ATG proteins; the LC3-II that is present on the concave surface remains associated with the completed autophagosome. [44][45][46] As a consequence, LC3-II has widely been used as an autophagic marker. 47,48 The final stage of autophagy is initiated by autophagosome-lysosome fusion, a process recently shown to involve interaction between STX17 (syntaxin 17) as an autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) with its targets SNAP29 and VAMP8, which drives the fusion between the outer autophagosomal membrane and the lysosomal membrane.…”

Structural and Functional Analysis of a Novel Interaction Motif within UFM1-activating Enzyme 5 (UBA5) Required for Binding to Ubiquitin-like Proteins and Ufmylation

“…In addition to its interaction with polyUb chains, p62/SQSTM1 interacts directly with the UBL proteins LC3/GABARAPs, which are conjugated to phosphatidylethanolamine enriched in autophagic membranes. Membrane-conjugated LC3/GABARAPs mediate lipid bilayer tethering and hemifusion (15,16), drive expansion of autophagosomes, and via autophagic receptors, target autophagy cargo to the endolysosomal compartment (17). p62/SQSTM1 binds LC3/GABARAPs via a short linear sequence, designated the LC3-interacting region (LIR) and broadly defined by the core sequence (W/F/Y)XX(L/I/V), where X is any amino acid (aa) (13,18).…”