Approaches to the Study of Atg8-Mediated Membrane Dynamics In Vitro

Jotwani, Anjali; Richerson, Diana; Motta, Isabelle; Julca-Zevallos, Omar; Melia, Thomas J.

doi:10.1016/b978-0-12-386487-1.00005-5

Cited by 15 publications

(15 citation statements)

References 62 publications

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“…This would be consistent with a portion of GL1-PE directly engaging the apposing bilayer via a membraneembedding motif as was first demonstrated by Elazar and colleagues 20 . However, this is also consistent with the possibility that 55% PE membranes support additional membrane dynamics including membrane fusion 18,19 . Indeed, on small extruded liposomes, we see that 55% PE supports fusion for GL1, GL2, and LC3 while 30% PE does not (Sup.…”

Section: Gabarap-dependent Tethering Of Small Liposomessupporting

confidence: 75%

“…This result has been reproduced by several groups 18,19 and extended to the mammalian and C. elegans homologs of Atg8, LC3 and GABARAP [18][19][20][21] . An intriguing possibility is that membrane tethering could be used either to recruit vesicles to the growing IM (to facilitate membrane growth) or to initiate closure of the autophagosome by pulling the edges of the dilating rim together.…”

Section: Introductionmentioning

confidence: 60%

“…To study the role of the Atg8 family independent of other cellular factors, the lipidation of Atg8 family proteins has also been reconstituted onto model membranes including small liposomes through the same ubiquitin-like reaction (e.g. 11,19,21,[27][28][29][30] , and Figure 1A). Light scattering measurements and phase microscopy show that formation of Atg8-PE and LC3-PE is correlated with a quick and robust aggregation of the liposomes, establishing a natural capacity in these proteins for membrane tethering 11,18,20,21 .…”

Section: Gabarap-dependent Tethering Of Small Liposomesmentioning

confidence: 99%

“…In vitro lipidation reactions were performed as previously described 18,19,28 . Each reaction is 150 uL in volume and contains purified Atg3 (2 μM), Atg7 (2 μM), Alexa488-GABARAPL1 (12-15 μM) or its homologs, mix with LUVs described above (2 mM lipid composed of 30% DOPE, 1% DOPE Atto647 or DOPE Rho, 69% DOPC or 68.5% DOPC + 0.5% DSPE-biotin), 1 mM dithiothreitol, and 1 mM ATP.…”

Section: In-vitro Reconstituted Lipidation Reactions To Decorate Gabamentioning

confidence: 99%

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GABARAP Like-1 enrichment on membranes: Direct observation of trans-homo-oligomerization between membranes and curvature-dependent partitioning into membrane tubules

Motta¹,

Nguyen

Gardavot³

et al. 2018

Preprint

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The Atg8/LC3/GABARAP protein family has been implicated in membrane remodeling events on the growing autophagosome. In particular, each of these proteins can form a protein-lipid conjugate that has been shown in vitro to drive liposome aggregation and in some cases membrane fusion. Furthermore, yeast Atg8 has been described as a curvature sensing protein, through its natural capacity to concentrate on highly curved membranes. A key advance with yeast Atg8, was the introduction of Giant Unilamellar Vesicles (GUVs) as an in vitro support that could allow membrane deformation and tethering to be observed by simple microscopy. Further, micromanipulation of an individual GUV could be used to create local areas of curvature to follow Atg8 partitioning. Here, we use a recently developed method to decorate GUVs with the mammalian Atg8 protein GABARAPL1 and establish the generality of the observations made on yeast Atg8. Then we apply double micromanipulation, the capture and positioning of two independently prepared GUVs, to test elements of the mechanism, speed and reversibility of mammalian Atg8 protein-mediated tethering. We find that the membranes adhere through GABARAPL1/GABARAPL1 homotypic trans-interactions. On a single membrane with two regions with significantly different curvatures we observed that the regions of higher curvature can be enriched up to 10 times in GABARAPL1 compared to the planar regions. This approach has the potential to allow the formation and study of specific topographically-controlled interfaces involving Atg8-proteins and their targets on apposing membranes.

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Section: Gabarap-dependent Tethering Of Small Liposomessupporting

confidence: 75%

Section: Introductionmentioning

confidence: 60%

Section: Gabarap-dependent Tethering Of Small Liposomesmentioning

confidence: 99%

Section: In-vitro Reconstituted Lipidation Reactions To Decorate Gabamentioning

confidence: 99%

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GABARAP Like-1 enrichment on membranes: Direct observation of trans-homo-oligomerization between membranes and curvature-dependent partitioning into membrane tubules

Motta¹,

Nguyen

Gardavot³

et al. 2018

Preprint

Self Cite

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show abstract

“…B) To test the impact of curvature on lipidation, we compared liposomes with unphysiologically high surface densities of PE (55 mole %) as have been used in other Atg8 or LC3 lipidation publications (e.g. 4,21,49 ), with liposomes having the highest densities of PE observed in mammalian organelle membranes (30 mole %). At physiologically relevant concentrations of DOPE (30 mole %), lipidation becomes membrane curvature dependent.…”

Section: Figurementioning

confidence: 99%

Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3

et al. 2014

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The protein biochemistry supporting autophagosome growth on the cup-like isolation membrane is likely different from the biochemistry on the closed and maturing autophagosome. Thus, the highly curved rim of the cup may serve as a functionally-required surface for transiently-associated components of the early-acting autophagic machinery. Here we demonstrate that the E2-like enzyme, Atg3, facilitates LC3/GABARAPlipidation only on membranes exhibiting local lipid-packing defects. This activity requires an amino-terminal amphipathic helix similar to motifs found on proteins targeting highly curved intracellular membranes. By tuning the hydrophobicity of this motif, we can promote or inhibit lipidation in vitro and in rescue experiments in Atg3 knockout cells, implying a physiologic role for this stress detection. The need for extensive lipid-packing defects suggests that Atg3 is designed to work at highly-curved membranes perhaps including the limiting edge of the growing phagophore.

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The coordination of membrane fission and fusion at the end of autophagosome maturation

Melia

2017

Current Opinion in Cell Biology

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The two major objectives of macroautophagy are to sequester cargo away from the cytoplasm and deliver this material for breakdown in the lysosome. Sequestration is complete when the autophagosome membrane undergoes fission to produce separate inner and outer membranes, while delivery into the lysosome requires fusion of the outer autophagosome membrane with the lysosome membrane. Thus, the merging of membranes through fission and fusion underlies each of the pivotal events in macroautophagic clearance. How these merging events are controlled in the cell is poorly understood. Several recent studies however suggest that the two events may be temporally coordinated and rely upon members of the classic membrane fusion SNARE family as well as the autophagy-specific family of Atg8 proteins.

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Approaches to the Study of Atg8-Mediated Membrane Dynamics In Vitro

Cited by 15 publications

References 62 publications

GABARAP Like-1 enrichment on membranes: Direct observation of trans-homo-oligomerization between membranes and curvature-dependent partitioning into membrane tubules

GABARAP Like-1 enrichment on membranes: Direct observation of trans-homo-oligomerization between membranes and curvature-dependent partitioning into membrane tubules

Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3

The coordination of membrane fission and fusion at the end of autophagosome maturation

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