In Vitro Assays of Lipidation of Mammalian Atg8 Homologs

Isei Tanida1, Takashi Ueno1, Eiki Kominami2

1 Laboratory of Proteomics and Biomolecular Science, Juntendo University Graduate School of Medicine, Tokyo, 2 Juntendo University Graduate School of Medicine, Tokyo
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.20
DOI:  10.1002/0471143030.cb1120s64
Online Posting Date:  September, 2014
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Abstract

Atg8 modifier in yeast is conjugated to phosphatidylethanolamine via ubiquitylation‐like reactions essential for autophagy. Mammalian Atg8 homologs (Atg8s) including LC3, GABARAP, and GATE‐16, are also ubiquitin‐like modifiers. The carboxyl termini of mammalian Atg8 homologs are cleaved by Atg4B, a cysteine protease, to expose carboxyl terminal Gly which is essential for this ubiquitylation‐like reaction. Thereafter, the Atg8 homologs are activated by Atg7, an E1‐like enzyme, to form unstable Atg7‐Atg8 E1‐substrate intermediates via a thioester bond. The activated Atg8 homologs are transferred to mammalian Atg3, an E2‐like enzyme, to form unstable Atg3‐Atg8 E2‐substrate intermediates via a thioester bond. Finally, Atg8 homologs are conjugated to phospholipids, phosphatidylethanolamine, and phosphatidylserine. Here, we describe a protocol for the reconstituted conjugation systems for mammalian Atg8 homologs in vitro using purified recombinant Atg proteins and liposomes. Curr. Protoc. Cell Biol. 64:11.20.1‐11.20.13. © 2014 by John Wiley & Sons, Inc.

Keywords: ubiquitin‐like modifier; autophagy; UBL; Atg8; GABARAP; GATE‐16; LC3

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Mammalian Atg Proteins Essential for In Vitro Assays of Lipidation of Mammalian Atg8 Homologs
  • Basic Protocol 2: In Vitro Assay of Formation of the Atg7‐Atg8 E1‐Substrate Intermediate
  • Basic Protocol 3: In Vitro Assay of Formation of the Atg3‐Atg8 E2‐Substrate Intermediate
  • Basic Protocol 4: In Vitro Assays of Lipidation of Mammalian Atg8 Homologs
  • Support Protocol 1: Preparation of Liposomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of Mammalian Atg Proteins Essential for In Vitro Assays of Lipidation of Mammalian Atg8 Homologs

  Materials
  • Escherichia coli BL21(DE3)
  • Plasmids (Sou et al., ):
    • pGEX‐6P‐hATG7 plasmid (for expression of human Atg7 in E. coli)
    • pGEX‐6P‐hATG3 plasmid (for expression of human Atg3 in E. coli)
    • pGEX‐6P‐hLC3TFG plasmid (for expression of human LC3 TFG in E. coli)
    • pGEX‐6P‐hLC3TF plasmid (for expression of human LC3 TF in E. coli)
    • pGEX‐6P‐hGABARAPVYG plasmid (for expression of human GABARAP VYG in E. coli)
    • pGEX‐6P‐hGABARAPVY plasmid (for expression of human GABARAP VY in E. coli)
    • pGEX‐6P‐hGATE‐16TFG plasmid (for expression of human GATE‐16 TFG in E. coli)
    • pGEX‐6P‐hGATE‐16TF plasmid (for expression of human GATE‐16 TF in E. coli)
  • LB agar plate containing 100 µg/ml ampicillin (see recipe)
  • LB medium containing 150 µg/ml ampicillin (see recipe)
  • 1 M isopropyl‐β‐D‐thiogalactopyranoside (IPTG; see recipe)
  • PBS (ice‐cold; see recipe)
  • SB solution (see recipe)
  • CB solution (see recipe)
  • TN buffer (see recipe)
  • 60% glycerol in TN buffer (see recipe)
  • Glutathione Sepharose (4B beads, GE Healthcare Life Sciences)
  • Prescission protease (GE Healthcare Life Sciences)
  • Centrifugal separator
  • Probe sonicator
  • Rotator
  • Low‐temperature incubator shaker

Basic Protocol 2: In Vitro Assay of Formation of the Atg7‐Atg8 E1‐Substrate Intermediate

  Materials
  • TN buffer (see recipe)
  • SDS solution (see recipe)
  • 1 M dithiothreitol (DTT) solution (prepare immediately before use)
  • 100 mM ATP
  • 1 M MgCl 2
  • Purified Atg proteins (see protocol 1)
  • Anti‐hAtg7 antibody (Cell Signaling Tech., cat. no. 2631 or Santa Cruz Biotechnology, cat. no. sc‐8668)
  • Anti‐LC3 antibody (Cell Signaling Tech., cat. no. 2775, 3868, or 12741)
  • Anti‐GABARAP antibody (MBL, cat. no. PM037)
  • Anti‐GATE‐16 antibody (MBL, cat. no. PM038)
  • HRP‐conjugated secondary antibodies (Jackson ImmunoResearch; anti‐rabbit IgG, cat. no. 111‐035‐144; anti‐mouse IgG, cat. no. 115‐035‐146; anti‐goat IgG, cat. no. 705‐035‐147)
  • SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific)
  • SDS‐PAGE equipment (e. g., Nu‐PAGE SDS‐GEL system, Life Technologies)
  • Immunoblotting equipment (e. g., TransBlot SD cell, BIO‐RAD)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher et al., ) and immunoblotting (Gallagher et al., )

Basic Protocol 3: In Vitro Assay of Formation of the Atg3‐Atg8 E2‐Substrate Intermediate

  Materials
  • TN buffer (see recipe)
  • SDS solution (see recipe)
  • 1 M dithiothreitol (DTT) solution (prepare immediately before use)
  • 100 mM ATP
  • 1 M MgCl 2
  • Purified Atg proteins (see protocol 1)
  • Anti‐hAtg3 antibody (Cell Signaling Tech., cat. no. 3415)
  • Anti‐hAtg7 antibody (Cell Signaling Tech., cat. no. 2631 or Santa Cruz Biotechnology, cat. no. sc‐8668)
  • Anti‐LC3 antibody (Cell Signaling Tech., cat. no. 2775, 3868, or 12741)
  • Anti‐GABARAP antibody (MBL, cat. no. PM037)
  • Anti‐GATE‐16 antibody (MBL, cat. no. PM038)
  • HRP‐conjugated secondary antibodies (Jackson ImmunoResearch; anti‐rabbit IgG, cat. no. 111‐035‐144; anti‐mouse IgG, cat. no. 115‐035‐146; anti‐goat IgG, cat. no. 705‐035‐147)
  • Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific)
  • SDS‐PAGE equipment (e. g., Nu‐PAGE SDS‐GEL system, Life Technologies)
  • Immunoblotting equipment (e. g., TransBlot SD cell, BIO‐RAD)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher et al., ) and immunoblotting (Gallagher et al., )

Basic Protocol 4: In Vitro Assays of Lipidation of Mammalian Atg8 Homologs

  Materials
  • Purified Atg proteins (see protocol 1)
  • Liposomes (see protocol 5Support Protocol)
  • TN buffer (see recipe)
  • SDS solution (see recipe)
  • 1 M dithiothreitol (DTT) solution (prepare immediately before use)
  • 100 mM ATP
  • 1 M MgCl 2
  • SDS‐PAGE equipment (e. g., Nu‐PAGE SDS‐GEL system, Life Technologies)
  • Immunoblotting equipment (e. g., TransBlot SD cell, BIO‐RAD)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher et al., ) and immunoblotting (Gallagher et al., )

Support Protocol 1: Preparation of Liposomes

  Materials
  • Lipid extracts, chloroform solution (e.g., Avanti Polar Lipids, cat. no. 171201 or prepare total lipids by the method of Bligh and Dyer [1959])
  • 2‐Oleoyl‐1‐palmitoyl‐sn‐glycero‐3‐phosphocholine (POPC), chloroform solution (Avanti Polar Lipids, cat. no. 850457)
  • 1, 2‐Dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE), chloroform solution (Avanti Polar Lipids, cat. no. 850725)
  • 1, 2‐Dioleoyl‐sn‐glycero‐3‐phospho‐L‐serine, sodium salt (DOPS), chloroform solution (Avanti Polar Lipids, cat. no. 840035)
  • TN buffer (see recipe)
  • PL buffer (see recipe)
  • Vacuum bell jars
  • Vacuum pump
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Figures

Videos

Literature Cited

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