doi: 10.1038/ncb2940.
Epub 2014 Apr 20.
Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3
Affiliations
- PMID: 24747438
- PMCID: PMC4111135
- DOI: 10.1038/ncb2940
Item in Clipboard
Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3
Nat Cell Biol.
2014 May.
Erratum in
- Nat Cell Biol. 2014 Aug;16(8):821
- Nat Cell Biol. 2014 Jul;16(7):716
Abstract
The components supporting autophagosome growth on the cup-like isolation membrane are likely to be different from those found on closed and maturing autophagosomes. 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/GABARAP lipidation 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.
Figures
A) Cartoon of the ubiquitin-like lipidation reaction of autophagy. B) Conjugation of LC3 and two other mammalian homologs (GL1 and GL2) with PE was performed essentially as described previously. Complete reactions contain 1.5 μM Atg7, 2.5 μM Atg3, 8-12μM LC3/GL1/GL2, 2 mM lipid (30nm liposomes composed of 10 mole% bl-PI, 55 mole% DOPE, 0.15 mole% Rh-DOPE and 34.85 mole% POPC), 1mM DTT and 1mM ATP and were run at 30°C for 90 min. Each reaction was run on a 12% SDS-PAGE gel and visualized by coomassie blue stain. The mobility of the lipidated protein is faster than the corresponding non-lipidated forms. Intermediates in the reaction are also detectable, including the Atg7-GRL1 conjugate, the Atg3-GRL1 conjugate, and a modified form of GRL1, GRL2 and LC3 that is labeled simply as GRL1/GRL2/LC3-intermediate and is likely the adenylated form of each LC3-related protein. C) The lipidation reactions are highly dependent upon the molar percentage of DOPE in the liposomes. Reactions are as in B, but run on 400 nm liposomes. Liposomes are composed of the indicated amount of DOPE, 10 mole% bl-PI, 0.15 mole% Rh-DOPE and POPC. D) Completed reactions in C were subjected to density gradient flotation to isolate only the lipidated material. Floated samples were run on SDS-PAGE and imaged by coomassie. Uncropped versions of all gels are included in the Supplemental Figure 7.
A) Cartoon depicting incompatibility of idealized cone-shaped lipids with planar assemblies. The spontaneous curvature in a lipid monolayer scales qualitatively with the idealized shape of the lipid where the idealized volume can be approximated as a cylinder (PC) or a cone (PE). When membrane curvature deviates significantly from the molecular spontaneous radius of curvature for an individual lipid, packing defects arise (i.e. , ) forcing lipids to adopt sub-optimal configurations, producing local monolayer stress (see ). Resolution of the local stress associated with these incompatibilities can include changes in lipid structure, a reorganization of lipid composition or the insertion of non-lipidic molecules including proteins. B) Filling the defects with inverted cone-shaped lipids inhibits lipidation. In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mole% DOPE (2mM total lipid) as in figure 1. To test whether inverted cones influenced the reaction, increasing amounts of Stearoyl CoA was added immediately before initiating the reaction with ATP. Quantification is of n=3 samples, error bars represent standard deviation. p-values represent a comparison to samples without stearyoyl-CoA. **: p<0.1; *:p<0.05. C) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction. In vitro coupling reactions of GL1 were run on liposomes of three different sizes (400nm and 50 nm extruded liposomes and sonicated liposomes) and of three different lipid compositions. Lipid compositions include 30 mole% DPPE, DSPE or DOPE, 10% bl-PI, and POPC. Total lipid = 2mM. Quantification is of n=3 samples, error bars represent standard deviation. The extent of lipidation in B and C is plotted as a percentage of total GL1 as determined by densitometry. p-values represent comparison to most active liposomes (sonicated DOPE). **: p<0.1; *:p<0.05.
A) Membrane defects resembling those accumulating with high concentrations of PE are prevalent in vivo at sites of high curvature and can be recapitulated in vitro by forming liposomes of different diameters. 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. ,,), 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. In vitro coupling reactions were performed as in Figure 1, except that liposomes were made by extrusion through membranes with different pore diameters (as indicated). Actual diameters of these liposomes were determined by dynamic light scattering (Table I). B-Top) The lipidation of GL1, GL2, and LC3 proceeds efficiently and with relatively little curvature dependence upon liposomes composed of 55% PE. However, when the PE is reduced to 30%, flat liposomes are no longer suitable for lipidation, while highly curved liposomes (100 nm diameter or less) continue to couple effectively. B-bottom) To confirm that this curvature dependence reveals the lipidated product and not the intermediate, the 30% reactions were each run on nycodenz flotation gradients. Only the lipidated product is recovered at the top of the gradient. C) The lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes. Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes (400 nm – black squares and sonicated – red circles) and varying DOPE mole%. The extent of lipidation is determined as in figure 2. (n=2 for 30 and 55% PE, n=4 for all the other points, error bars represent standard deviation). p-values were determined comparing sonicated to 400nm samples at each PE concentration.**: p<0.1; *:p<0.01. Uncropped versions of all gels are included in the Supplemental Figure 7.
A) The accumulation of the Atg3-GL1 conjugate depends upon the extent of membrane curvature. In vitro GL1 coupling reactions were run as in figure 1. The reactions were visualized by immunoblot against Atg3 (top) and by coomassie staining of GRL1 (bottom). The accumulation of Atg3-GL1 is inversely related to the formation of the lipidated GRL1 suggesting that the membrane curvature-dependent step may involve Atg3 recognition of the membrane surface. B) Atg3 binding to liposomes. Atg3 (10 μM) was incubated at 30°C for 90 min with 0, 30, or 55 mole% DOPE containing liposomes (5 mM lipids) of varying sizes (extrusion membrane dimensions are shown on the figure, actual final sizes were determined by dynamic light scattering (Table I)). The liposome associated Atg3 was recovered by nycodenz density gradient centrifugation and analysed by SDS PAGE. C) Quantification of liposome-associated Atg3. Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in B. Error bars indicate SEM from three independent experiments. p-values calculated as compared to putative negative control (0% PE, 400 nm). *:p< 0.05. Uncropped versions of all gels are included in the Supplemental Figure 7.
A) Helical wheel representation of the predicted N-terminal amphipathic helix (aa 4-26) of mouse Atg3 (wheel generated in Heliquest). B) Analysis of reaction efficiency for point mutants of the amphipathic helix. GL1 lipidation reactions were run on 400nm and sonicated liposomes (30 mole% DOPE) with the indicated Atg3 mutants. The extent of GL1-PE formation (top gel) was determined from densitometry and is plotted as percent of total GL1 (bar graph). The ability to form the Atg3-GL1 conjugate was also assessed for each mutant (bottom immunoblot). Note that this conjugate forms in every case, indicating that no mutant inhibited a step upstream in the reaction. Further, this conjugate specifically accumulates in reactions where lipidation is impaired. C) Curvature dependence of K11L mutant binding to liposomes compared to wild-type Atg3. The binding reaction was performed as in Figure 4b on 30DOPE% liposomes. F = Float-up; T = Total Input. D) K11 represents a tunable node through which curvature dependence of the lipidation reaction can be controlled. Lipidation assays were run as in B. Total GL1 lipidation was assessed from reactions with wildtype Atg3 or mutant forms of Atg3 in which position K11 was changed as indicated. Lipidation efficiency on low PE/low curvature liposomes (30% PE, 400 nm) increases with increasing hydrophobicity of the new amino acid, while liposomes with high PE (55%) or high curvature (sonicated) are good substrates for all active forms of Atg3. D-left) coomassie gels of individual lipidation reactions. D-right) densitometry of coomassie-stained gels revealed the ratio of GL1-PE to the sum of all three GL1 species as in figure 2. Light grey bars are sonicated liposomes; dark grey bars are 400 nm extruded liposomes. S = Sonicated liposomes; 400 = 400nm liposomes. For b, c, p-values were calculated between sonicated and 400 nm samples of the same mutant. For d, p-values represent comparison to WT protein on the same liposome size and composition. For all panels, n=3 independent experiments, error bars represent standard deviation and**: p< 0.05; *:p< 0.01. Uncropped gels in Supplemental Figure 7.
Atg3 knockout MEFs (Atg3 -/-) are incapable of forming LC3-II and do not accumulate LC3 puncta. Instead, they accumulate autophagic intermediates that are positive for Atg16. To test if the amphipathic helix is critical for in vivo function, Atg3 with wild-type or mutant forms of the amphipathic helix was introduced into Atg3 -/- MEFs by lentiviral infection and rescue of the three phenotypes was tested. A) Immunoblot analysis of LC3-II, GL2-II and GABARAP-II formation. GABARAP lipidation is only significantly observed in the presence of Bafilomycin A1 (BafA1: 100 nM). Asterisk indicates a non-specific band recognized by the Atg3 antibody. B) Cells were infected as in A and transduced cells were selected with puromycin for 1-2 days before starvation and immunolabeling with the anti-LC3B antibody. Representative images are shown in B. C) The number of LC3 puncta per cell was counted manually from 22-96 cells per replicate. Raw data can be found in Supplemental Figure 5. p-values represent difference from vector negative control. n = 2-5 independent experiments; the precise n values and number of cells per experiment are listed in the Methods. D) Cells as in B were kept in full media before being subjected to fixation and immunolabeling with the anti-Atg16L antibody. Atg16L puncta area as a fraction of total cellular area was obtained as described in Methods. This ratio was plotted as arbitrary units. Grey dashed line indicates the value for Atg3-/- cells rescued with wild-type Atg3. n = 2 (for V15K) or 3 (for all others) independent experiments; the precise n values and number of fields visualised per experiment are listed in the Methods. Raw data can be found in Supplemental Figure 4. Atg3 variants that support lipidation of LC3 family proteins in vitro (Figure 5) are indicated with black bars, while those that do not support lipidation are indicated with white bars. p-values represent difference from wild-type rescue.**:p<0.1; *: p<0.05. Uncropped versions of all gels are included in the Supplemental Figure 7.
Comment in
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Selective autophagy goes exclusive.Nat Cell Biol. 2014 May;16(5):395-7. doi: 10.1038/ncb2961. Nat Cell Biol. 2014. PMID: 24914435
References
-
- Ichimura Y, et al. A ubiquitin-like system mediates protein lipidation. Nature. 2000;408:488–492. - PubMed
-
- Kabeya Y, et al. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. Journal of cell science. 2004;117:2805–2812. - PubMed
-
- Nakatogawa H, Ichimura Y, Ohsumi Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell. 2007;130:165–178. - PubMed
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