通过分析ATG8脂化解析拟南芥的自噬活性

Mengqian Luo; Xiaohong  Zhuang

doi:10.21769/BioProtoc.2880

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Peer-reviewed

Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana

通过分析ATG8脂化解析拟南芥的自噬活性

ML Mengqian Luo

XZ Xiaohong Zhuang email

发布: 2018年06月20日第8卷第12期 DOI: 10.21769/BioProtoc.2880 浏览次数: 7859

评审: Zhibing LaiYasin DagdasAnonymous reviewer(s)

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Cover of Proceedings of the National Academy of Sciences of the United States of America, featuring study using the protocol.

Jan 2017

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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.

Keywords: ATG8 (ATG8)

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

10 µl pipette tips (Thermo Fisher Scientific, catalog number: 3510 )
200 µl pipette tips (Wolf Laboratories, catalog number: 2100.YN )
1,000 µl pipette tips (Thermo Fisher Scientific, catalog number: 3580 )
1.5 ml microcentrifuge tubes (Corning, Axygen^®, catalog number: MCT-150-C )
PVDF membrane
X-ray film (Advansta, catalog number: L-07014-100 )
5-day-old seedlings
MS salt (Caisson, catalog number: MSP01-50LT )
UltraPure^TM Sucrose (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15503022 )
BTH (Acibenzolar-S-methyl) (Sigma-Aldrich, catalog number: 32820 )
Methanol (VWR, BDH, catalog number: 10158 )
Liquid nitrogen
PIC (Protease Inhibitor Mixture) (Roche Diagnostics, catalog number: 11873580001 )
Tris Base (Caisson, catalog number: T041-1KG )
NaCl (Alfa Aesar, USB, catalog number: J21618 )
EDTA (Alfa Aesar, USB, catalog number: J15701 )
SDS (Sodium dodecyl sulfate) (Alfa Aesar, USB, catalog number: J75819 )
Triton X-100 (GE Healthcare, Amerrsham, catalog number: 17-1315-01 )
Glycerol ultrapure (Alfa Aesar, USB, catalog number: J16374 )
Bromophenol Blue (Sigma-Aldrich, catalog number: B5525 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Urea (Alfa Aesar, USB, catalog number: J75826 )
40% Acrylamide/Bis Solution (Bio-Rad Laboratories, catalog number: 161-0148 )
TEMED (Bio-Rad Laboratories, catalog number: 161-0801 )
APS (Ammonium persulfate) (USB, catalog number: US76322 )
Non-fat milk powder
ATG8 antibody (Agrisera, catalog number: AS14 2769 )
cFBPase (Agrisera, catalog number: AS04 043 )
Secondary antibody (Anti-rabbit IgG peroxidase conjugate, Sigma-Aldrich, catalog number: A6154 )
Precision Plus Protein^TM Dual Color Standards (Bio-Rad Laboratories, catalog number: 161-0374 )
Sodium hydrogen carbonate (NaHCO₃) (VWR, catalog number: 144-55-8 )
Sodium carbonate (Na₂CO₃) (USB, catalog number: 21602 )
Sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O) (USB, catalog number: 20233 )
Sodium phosphate dibasic dihydrate (Na₂HPO₄·2H₂O) (Sigma-Aldrich, catalog number: 04272 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 31248 )
Tween-20 (Sigma-Aldrich, catalog number: 63158 )
MS liquid medium (see Recipes)
10 mM BTH stock (see Recipes)
25x PIC (see Recipes)
10% (v/v) Triton X-100 (see Recipes)
1 M Tris-HCl stock (pH 6.8 or pH 7.4) (see Recipes)
0.5 M EDTA (pH 8.0) (see Recipes)
5x extraction buffer (see Recipes)
1x extraction buffer containing 1x PIC and 1% (v/v) Triton X-100 (see Recipes)
5x sample loading dye (see Recipes)
30% APS (see Recipes)
3x separation buffer (see Recipes)
5x stacking buffer (see Recipes)
15% SDS-PAGE gel with 6 M urea (see Recipes)
1. 15% Urea separating gel
2. 5% Stacking gel
Running buffer (see Recipes)
Transfer buffer (see Recipes)
PBS (see Recipes)
PBS-T (see Recipes)

Equipment

Eppendorf Research^® plus Pipette 0.5-10 µl (Eppendorf, catalog number: 3120000020 )
Eppendorf Research^® plus Pipette 10-100 µl (Eppendorf, catalog number: 3120000046 )
Eppendorf Research^® plus Pipette 100-1,000 µl (Eppendorf, catalog number: 3120000062 )
Mortar and pestle
Centrifuge (Eppendorf, model: 5430 )
X-ray film cassette (Amersham Biosciences Hypercassette^TM)
Ultracentrifuge tube (7 x 20 mm)
Ultracentrifuge (Beckman Coulter, model: Optima^TM MAX-XP )
Western Blotting apparatus (Bio-Rad)
Developer machine (Fujifilm FPM100A)

Procedure

English

中文翻译

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如何引用

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.