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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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Reconstitution of eGFP-Atg8 conjugation using GUVs. (A) GUVs incubated with monomeric eGFP-Atg8 in the presence of Atg3, Atg7 and the Atg12–Atg5-Atg16 complex. On the left ATP was added to the reaction. On the right ATP was omitted. The fluorescence signal of eGFP-Atg8 is shown in false color (spectrum). (B) Same reaction as in (A) right panel but here the membrane of the GUV is labeled by incorporation of Marina blue PE. For the conjugation reaction the proteins were used at the final concentrations of 80 nM (Atg3 and Atg7) and 400 nM (eGFP-Atg8 and Atg12–Atg5-Atg16). The reaction was performed in the presence or absence of 1 mM ATP. Scale bars: 5 μm.
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f0020: Reconstitution of eGFP-Atg8 conjugation using GUVs. (A) GUVs incubated with monomeric eGFP-Atg8 in the presence of Atg3, Atg7 and the Atg12–Atg5-Atg16 complex. On the left ATP was added to the reaction. On the right ATP was omitted. The fluorescence signal of eGFP-Atg8 is shown in false color (spectrum). (B) Same reaction as in (A) right panel but here the membrane of the GUV is labeled by incorporation of Marina blue PE. For the conjugation reaction the proteins were used at the final concentrations of 80 nM (Atg3 and Atg7) and 400 nM (eGFP-Atg8 and Atg12–Atg5-Atg16). The reaction was performed in the presence or absence of 1 mM ATP. Scale bars: 5 μm.

Mentions: The following lipid mix was used: 40% DOPC, 35% DOPS, 20% DOPE, 5% PI3P. 2% Marina Blue (Invitrogen) at the expense of DOPC was added to the membrane labeled GUVs shown in Fig. 4. All lipids were purchased from Avanti Polar Lipids Inc. Marina blue PE was from Invitrogen.


In vitro systems for Atg8 lipidation.

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

Reconstitution of eGFP-Atg8 conjugation using GUVs. (A) GUVs incubated with monomeric eGFP-Atg8 in the presence of Atg3, Atg7 and the Atg12–Atg5-Atg16 complex. On the left ATP was added to the reaction. On the right ATP was omitted. The fluorescence signal of eGFP-Atg8 is shown in false color (spectrum). (B) Same reaction as in (A) right panel but here the membrane of the GUV is labeled by incorporation of Marina blue PE. For the conjugation reaction the proteins were used at the final concentrations of 80 nM (Atg3 and Atg7) and 400 nM (eGFP-Atg8 and Atg12–Atg5-Atg16). The reaction was performed in the presence or absence of 1 mM ATP. Scale bars: 5 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0020: Reconstitution of eGFP-Atg8 conjugation using GUVs. (A) GUVs incubated with monomeric eGFP-Atg8 in the presence of Atg3, Atg7 and the Atg12–Atg5-Atg16 complex. On the left ATP was added to the reaction. On the right ATP was omitted. The fluorescence signal of eGFP-Atg8 is shown in false color (spectrum). (B) Same reaction as in (A) right panel but here the membrane of the GUV is labeled by incorporation of Marina blue PE. For the conjugation reaction the proteins were used at the final concentrations of 80 nM (Atg3 and Atg7) and 400 nM (eGFP-Atg8 and Atg12–Atg5-Atg16). The reaction was performed in the presence or absence of 1 mM ATP. Scale bars: 5 μm.
Mentions: The following lipid mix was used: 40% DOPC, 35% DOPS, 20% DOPE, 5% PI3P. 2% Marina Blue (Invitrogen) at the expense of DOPC was added to the membrane labeled GUVs shown in Fig. 4. All lipids were purchased from Avanti Polar Lipids Inc. Marina blue PE was from Invitrogen.

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