Published: Vol 8, Iss 12, Jun 20, 2018 DOI: 10.21769/BioProtoc.2880 Views: 7775
Reviewed by: Zhibing LaiYasin DagdasAnonymous reviewer(s)
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Abstract
As a fundamental metabolic pathway to degrade and recycle cellular cargos, autophagy is highly induced upon stress, starvation and senescence conditions in plants. A double-membrane structure named autophagosome will form during this process for cargo sequestration and delivery into the vacuole.
A number of regulators have been characterized in plants, including the autophagy-related (ATG) proteins and other plant-specific proteins. Among them, ATG8 will undergo a lipidation process to become a membrane-bound ATG8-phosphatidylethanolamine form and mark the growing autophagosomal membrane as well as the completed autophagosome. Therefore, ATG8 has been regarded as a marker for autophagosomes; and biochemical detection of the membrane-associated form of ATG8 is used as one of the principal methods for measurement of autophagic activity. Here, we describe an ATG8 lipidation assay for detection of the ATG8-PE form using Arabidopsis thaliana seedlings.
Background
Autophagy is an essential metabolic process which mediates the bulk degradation of the damaged organelles and unwanted cellular contents. During autophagy, a double-membrane structure called autophagosome will form and deliver the cargos into the vacuole for degradation. The autophagy-related (ATG) proteins are required to regulate the autophagic activity (Liu and Bassham, 2012). Among them, two conjugation systems, including ATG8 conjugate and ATG5-ATG12 conjugate, are involved for autophagosome formation. Upon autophagic induction, the ATG5-ATG12 conjugate forms and functions as an E3-like enzyme to promote ATG8 lipidation for binding to the phosphatidylethanolamine (PE) on the autophagosome membrane (Ohsumi, 2001). Although ATG8-PE on the outer membrane will be recycled before the autophagosome fusion with the vacuole, ATG8-PE on inner membrane will traffic together with the cargo into the vacuole for degradation. Thus, the amount of ATG8-PE usually correlates with the number of punctate ATG8-positive structures as well as autophagic activity (Mizushima et al., 2010). Particularly, due to the high hydrophobicity of ATG8-PE, ATG8-PE migrates faster than ATG8 in SDS-PAGE gel, though the actual molecular weight of ATG8-PE is larger than the unconjugated ATG8 (Mizushima and Yoshimori, 2007). Accordingly, the amount of ATG8-PE from cell membrane fraction (CM) can be detected by immunoblotting with ATG8 antibodies. For example, in Arabidopsis atg5 mutant, the level of ATG8-PE is severely impaired upon autophagic induction, whereas no autophagosome structures labeled by ATG8 are formed (Chung et al., 2010). Therefore, biochemical detection of the ATG8 lipidation can serve as a useful method to access the autophagic activity when combined with different treatments, which has been applied in our previous study as well as others related to plant autophagy (Chung et al., 2010; Suttangkakul et al., 2011; Li et al., 2014; Zhuang et al., 2017). Here, we describe the protocol for ATG8 lipidation detection by ultracentrifuge separation of the membrane and cytosol fractions using acibenzolar-S-methyl (BTH)-treated seedlings (Zhuang et al., 2017).
Materials and Reagents
Equipment
Procedure
Data analysis
As shown in Figure 1, in the wild-type (WT) background, the ATG8-PE from the CM sample increases after BTH induction, with a size about 12-15 kDa, indicating that autophagy is induced with the formation of autophagosomes. However, ATG8-PE is not detected in the ATG5 deficient mutant after autophagic induction, implying that autophagosome formation is inhibited. Differently, a higher level of ATG8-PE was detected in atg9 mutant, suggesting that autophagosome formation might be interrupted at a certain stage. There might be non-specific bands in the ATG8 antibody detection. Also, it should be pointed out that there are multiple isoforms of ATG8 in the Arabidopsis genome with different SDS-PAGE mobilities, resulting the detection of cross-reacting species with similar size to the ATG8-PE adducts and making the results contradictory (Chung et al., 2010). Therefore, it is critical to include both the WT and atg5 samples as the positive and negative controls respectively to identify the correct size of ATG8-PE, as atg5 mutant lacks ATG8-PE but accumulates a large amount of non-lipidated ATG8. Our further examinations under confocal and electron microscopy identified that abnormal tubules labeled by ATG8 accumulated in the atg9 mutant, thus demonstrating that ATG9 is required for autophagosome progression (Zhuang et al., 2017). Therefore, the ATG8 lipidation assay should combine with other approaches such as microscopy analysis to further assess autophagic activity.
Figure 1. ATG8 lipidation detection in Arabidopsis thaliana wild-type and atg mutants. The 5-day-old wild-type, atg5 and atg9 seedlings were incubated in the MS liquid medium with or without BTH treatment (+B and -B) for 8 hours respectively. Crude extracts were subjected to ultracentrifuge to collect the cell membrane fraction (CM), followed by immunoblotting with plant ATG8 antibody. cFBPase represents the loading control with immunoblotting cFBPase antibodies.
Notes
Recipes
Components | Volume/Quantity |
MS salt | 1.515 g |
Sucrose | 3.5 g |
ddH2O | up to 350 ml |
Components | Volume/Quantity |
BTH | 0.021 g |
Methanol | 10 ml |
Components | Volume/Quantity |
PIC | one tablet |
ddH2O | 2 ml |
Components | Volume/Quantity |
Triton X-100 | 10 ml |
ddH2O | 90 ml |
Components | Volume/Quantity |
Tris Base | 12.114 g |
ddH2O | to 100 ml |
Components | Volume/Quantity |
EDTA | 16.81 g |
ddH2O | 80 ml |
Components | Volume/Quantity |
250 mM Tris-HCl (pH 7.4) | 2.5 ml of 1 M Tris-HCl (pH 7.4) |
750 mM NaCl | 0.438 g |
5 mM EDTA | 0.1 ml of 0.5 M stock |
ddH2O | to 10 ml |
Components | Volume/Quantity |
1x extraction buffer | 2.5 ml of 5x extraction buffer |
1x PIC | 0.40 ml of 25x PIC |
1% (v/v) Triton X-100 | 1 ml of 10% (v/v) Triton X-100 |
ddH2O | to 10 ml |
Components | Volume/Quantity |
1 M Tris-HCl (pH 6.8) | 12.5 ml |
SDS | 5 g |
Glycerol | 25 ml |
Bromophenol Blue | 0.25 g |
β-mercaptoethanol | 6.25 ml |
ddH2O | to 50 ml |
Components | Volume/Quantity |
APS | 3 g |
ddH2O | 10 ml |
Components | Volume/Quantity |
Tris base | 136.2 g |
SDS | 3 g |
ddH2O | up to 1 L |
Components | Volume/Quantity |
Tris base | 37.85 g |
SDS | 2.5 g |
ddH2O | up to 500 ml |
Components | Volume/Quantity |
Urea | 3.6036 g |
Acry-bis (40%) | 3.75 ml |
3x separation buffer | 3.33 ml |
APS (30%) | 20 μl |
TEMED | 5 μl |
ddH2O | up to 10 ml |
Components | Volume/Quantity |
Acry-bis (40%) | 0.625 ml |
5x stacking buffer | 1 ml |
APS (30%) | 20 μl |
TEMED | 5 μl |
ddH2O | up to 5 ml |
Components | Volume/Quantity |
Tris Base | 30.3 g |
Glycine | 144 g |
SDS | 10 g |
ddH2O | up to 1 L |
Components | Volume/Quantity |
NaHCO3 | 8.4 g |
Na2CO3 | 3.2 g |
ddH2O | up to 1 L |
Components | Volume/Quantity |
NaCl | 80 g |
NaH2PO4·H2O | 2.3 g |
Na2HPO4·2H2O | 13.9 g |
KCl | 2 g |
ddH2O | up to 2 L |
Components | Volume/Quantity |
1x PBS | 1 L |
Tween-20 | 0.5 ml |
Acknowledgments
This protocol was adapted from Zhuang et al., 2017. Fundings from the Research Grants Council of Hong Kong (G-CUHK402/15, G-CUHK403/17, CUHK14130716, CUHK14102417, C4011-14R, C4012-16E, C4002-17G and AoE/M-05/12) and the National Natural Science Foundation of China (31470294 and 31670179) support this work.
Competing interests
The authors declare no conflicts of interests.
References
Article Information
Copyright
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Luo, M. and Zhuang, X. (2018). Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana. Bio-protocol 8(12): e2880. DOI: 10.21769/BioProtoc.2880.
Category
Plant Science > Plant immunity > Host-microbe interactions
Molecular Biology > Protein > Detection
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