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In vitro systems for Atg8 lipidation.

Zens B, Sawa-Makarska J, Martens S - Methods (2014)

Bottom Line: Among these are the components of two ubiquitin-like conjugation reactions that collectively mediate the conjugation of the ubiquitin-like Atg12 to the Atg5 protein and of the ubiquitin-like protein Atg8 to the headgroup of the membrane lipid phosphatidylethanolamine.The lipidated form of Atg8 is membrane-bound and marks the growing autophagosomal membrane as well as the completed autophagosome.The assays enable the study of the biochemical mechanisms of action of the Atg8 lipidation machinery and to analyze the impact of mutations and post-translational modifications of the conjugation machinery on Atg8 lipidation.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.

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In vitro Atg8 lipidation reaction using SUVs. The figure shows a 16% urea SDS page gel of an overnight Atg8 lipidation reaction. The conversion of Atg8 to Atg8–PE is detected by a characteristic size shift towards a faster migration behavior in this gel system. The addition of 6 M urea to the separating gel greatly facilitates the detection of the lipidated form of yeast Atg8. Lipidation of Atg8 occurs only in the presence of Atg3, Atg7, SUVs (derived from the E. coli lipids in the reaction shown) and ATP plus MgCl2 (all components). The Atg8–Atg3 intermediate can also be detected on this gel. The faster migrating Atg8 band in the absence of liposomes presumably represents the Atg8–AMP product formed by Atg7 [34]. The numbers on the left of the gel indicate the molecular weights of the marker bands in kDa.
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f0010: In vitro Atg8 lipidation reaction using SUVs. The figure shows a 16% urea SDS page gel of an overnight Atg8 lipidation reaction. The conversion of Atg8 to Atg8–PE is detected by a characteristic size shift towards a faster migration behavior in this gel system. The addition of 6 M urea to the separating gel greatly facilitates the detection of the lipidated form of yeast Atg8. Lipidation of Atg8 occurs only in the presence of Atg3, Atg7, SUVs (derived from the E. coli lipids in the reaction shown) and ATP plus MgCl2 (all components). The Atg8–Atg3 intermediate can also be detected on this gel. The faster migrating Atg8 band in the absence of liposomes presumably represents the Atg8–AMP product formed by Atg7 [34]. The numbers on the left of the gel indicate the molecular weights of the marker bands in kDa.

Mentions: SUVs were prepared using an E. coli total extract (Avanti Polar Lipids, Inc., 100500C), which contains between 55% and 60% PE (weight/weight) for the assays shown in Figs. 2 and 3A. For the Atg4 deconjugation assay shown in Fig. 3B the SUVs were composed of 40% POPC, 35% POPS, 20% POPE, 5% PI3P. 100 μl of the lipid stock (10 mg/ml) were transferred into a glass vial and dried under an argon stream. The dried lipids were further dried for an additional hour in a desiccator. Subsequently, the dried lipids were incubated with 1 ml of 50 mM HEPES, pH 7.5, 0.2 mM DTT (or 1 mM DTT for Fig. 3B) for 15 min. The lipids were resuspended by tapping and gently sonicated for 2 min in a water bath sonicator. The resuspended SUVs were then extruded 21 times through 0.4 μm membrane followed by extrusion through a 0.1 μm membrane (Whatman, Nucleopore) using the Mini Extruder from Avanti Polar Lipids Inc. The final SUVs suspension has a concentration of 1 mg lipids/ml buffer. SUVs are stable for 1–2 days when stored at 4 °C.


In vitro systems for Atg8 lipidation.

Zens B, Sawa-Makarska J, Martens S - Methods (2014)

In vitro Atg8 lipidation reaction using SUVs. The figure shows a 16% urea SDS page gel of an overnight Atg8 lipidation reaction. The conversion of Atg8 to Atg8–PE is detected by a characteristic size shift towards a faster migration behavior in this gel system. The addition of 6 M urea to the separating gel greatly facilitates the detection of the lipidated form of yeast Atg8. Lipidation of Atg8 occurs only in the presence of Atg3, Atg7, SUVs (derived from the E. coli lipids in the reaction shown) and ATP plus MgCl2 (all components). The Atg8–Atg3 intermediate can also be detected on this gel. The faster migrating Atg8 band in the absence of liposomes presumably represents the Atg8–AMP product formed by Atg7 [34]. The numbers on the left of the gel indicate the molecular weights of the marker bands in kDa.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358837&req=5

f0010: In vitro Atg8 lipidation reaction using SUVs. The figure shows a 16% urea SDS page gel of an overnight Atg8 lipidation reaction. The conversion of Atg8 to Atg8–PE is detected by a characteristic size shift towards a faster migration behavior in this gel system. The addition of 6 M urea to the separating gel greatly facilitates the detection of the lipidated form of yeast Atg8. Lipidation of Atg8 occurs only in the presence of Atg3, Atg7, SUVs (derived from the E. coli lipids in the reaction shown) and ATP plus MgCl2 (all components). The Atg8–Atg3 intermediate can also be detected on this gel. The faster migrating Atg8 band in the absence of liposomes presumably represents the Atg8–AMP product formed by Atg7 [34]. The numbers on the left of the gel indicate the molecular weights of the marker bands in kDa.
Mentions: SUVs were prepared using an E. coli total extract (Avanti Polar Lipids, Inc., 100500C), which contains between 55% and 60% PE (weight/weight) for the assays shown in Figs. 2 and 3A. For the Atg4 deconjugation assay shown in Fig. 3B the SUVs were composed of 40% POPC, 35% POPS, 20% POPE, 5% PI3P. 100 μl of the lipid stock (10 mg/ml) were transferred into a glass vial and dried under an argon stream. The dried lipids were further dried for an additional hour in a desiccator. Subsequently, the dried lipids were incubated with 1 ml of 50 mM HEPES, pH 7.5, 0.2 mM DTT (or 1 mM DTT for Fig. 3B) for 15 min. The lipids were resuspended by tapping and gently sonicated for 2 min in a water bath sonicator. The resuspended SUVs were then extruded 21 times through 0.4 μm membrane followed by extrusion through a 0.1 μm membrane (Whatman, Nucleopore) using the Mini Extruder from Avanti Polar Lipids Inc. The final SUVs suspension has a concentration of 1 mg lipids/ml buffer. SUVs are stable for 1–2 days when stored at 4 °C.

Bottom Line: Among these are the components of two ubiquitin-like conjugation reactions that collectively mediate the conjugation of the ubiquitin-like Atg12 to the Atg5 protein and of the ubiquitin-like protein Atg8 to the headgroup of the membrane lipid phosphatidylethanolamine.The lipidated form of Atg8 is membrane-bound and marks the growing autophagosomal membrane as well as the completed autophagosome.The assays enable the study of the biochemical mechanisms of action of the Atg8 lipidation machinery and to analyze the impact of mutations and post-translational modifications of the conjugation machinery on Atg8 lipidation.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.

Show MeSH
Related in: MedlinePlus