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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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Related in: MedlinePlus

In vitro Atg8 lipidation/de-lipidation reaction using SUVs. (A) Two Coomassie-stained gels of two time series of Atg8 conjugation reactions using SUVs derived from the E. coli lipids. The first reaction was conducted in the absence (left) and the second reaction was conducted in the presence (right) of the Atg12–Atg5-Atg16 complex. The lower gels shown in the bottom were run at lower polyacrylamide concentrations (12% as opposed to 16% for the upper two gels) in order to separate the Atg7 protein from the Atg12–Atg5 conjugate. Note the massive stimulation of Atg8 lipidation in the presence of the Atg12–Atg5-Atg16 complex. The numbers between the gels indicate the molecular weights of the marker bands in kDa. (B) Coomassie-stained gel of an Atg8 lipidation/de-lipidation reaction. The SUVs were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. Atg8 was first completely lipidated in the presence of Atg3, Atg7, the Atg12–Atg5 conjugate and ATP and MgCl2. After 30 min Atg4 and EDTA were added to final concentrations of 0.07 μM and 2 mM, respectively. After 2 min Atg8 was completely de-lipidated. The numbers to the left of the gel indicate the molecular weights of the marker bands in kDa. Note that the 35 kDa and 15 kDa bands separate into two bands in the presence of 6 M urea. (M: marker).
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f0015: In vitro Atg8 lipidation/de-lipidation reaction using SUVs. (A) Two Coomassie-stained gels of two time series of Atg8 conjugation reactions using SUVs derived from the E. coli lipids. The first reaction was conducted in the absence (left) and the second reaction was conducted in the presence (right) of the Atg12–Atg5-Atg16 complex. The lower gels shown in the bottom were run at lower polyacrylamide concentrations (12% as opposed to 16% for the upper two gels) in order to separate the Atg7 protein from the Atg12–Atg5 conjugate. Note the massive stimulation of Atg8 lipidation in the presence of the Atg12–Atg5-Atg16 complex. The numbers between the gels indicate the molecular weights of the marker bands in kDa. (B) Coomassie-stained gel of an Atg8 lipidation/de-lipidation reaction. The SUVs were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. Atg8 was first completely lipidated in the presence of Atg3, Atg7, the Atg12–Atg5 conjugate and ATP and MgCl2. After 30 min Atg4 and EDTA were added to final concentrations of 0.07 μM and 2 mM, respectively. After 2 min Atg8 was completely de-lipidated. The numbers to the left of the gel indicate the molecular weights of the marker bands in kDa. Note that the 35 kDa and 15 kDa bands separate into two bands in the presence of 6 M urea. (M: marker).

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/de-lipidation reaction using SUVs. (A) Two Coomassie-stained gels of two time series of Atg8 conjugation reactions using SUVs derived from the E. coli lipids. The first reaction was conducted in the absence (left) and the second reaction was conducted in the presence (right) of the Atg12–Atg5-Atg16 complex. The lower gels shown in the bottom were run at lower polyacrylamide concentrations (12% as opposed to 16% for the upper two gels) in order to separate the Atg7 protein from the Atg12–Atg5 conjugate. Note the massive stimulation of Atg8 lipidation in the presence of the Atg12–Atg5-Atg16 complex. The numbers between the gels indicate the molecular weights of the marker bands in kDa. (B) Coomassie-stained gel of an Atg8 lipidation/de-lipidation reaction. The SUVs were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. Atg8 was first completely lipidated in the presence of Atg3, Atg7, the Atg12–Atg5 conjugate and ATP and MgCl2. After 30 min Atg4 and EDTA were added to final concentrations of 0.07 μM and 2 mM, respectively. After 2 min Atg8 was completely de-lipidated. The numbers to the left of the gel indicate the molecular weights of the marker bands in kDa. Note that the 35 kDa and 15 kDa bands separate into two bands in the presence of 6 M urea. (M: marker).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0015: In vitro Atg8 lipidation/de-lipidation reaction using SUVs. (A) Two Coomassie-stained gels of two time series of Atg8 conjugation reactions using SUVs derived from the E. coli lipids. The first reaction was conducted in the absence (left) and the second reaction was conducted in the presence (right) of the Atg12–Atg5-Atg16 complex. The lower gels shown in the bottom were run at lower polyacrylamide concentrations (12% as opposed to 16% for the upper two gels) in order to separate the Atg7 protein from the Atg12–Atg5 conjugate. Note the massive stimulation of Atg8 lipidation in the presence of the Atg12–Atg5-Atg16 complex. The numbers between the gels indicate the molecular weights of the marker bands in kDa. (B) Coomassie-stained gel of an Atg8 lipidation/de-lipidation reaction. The SUVs were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. Atg8 was first completely lipidated in the presence of Atg3, Atg7, the Atg12–Atg5 conjugate and ATP and MgCl2. After 30 min Atg4 and EDTA were added to final concentrations of 0.07 μM and 2 mM, respectively. After 2 min Atg8 was completely de-lipidated. The numbers to the left of the gel indicate the molecular weights of the marker bands in kDa. Note that the 35 kDa and 15 kDa bands separate into two bands in the presence of 6 M urea. (M: marker).
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