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Studies of the Ubiquitin Proteasome System

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Abstract
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Abstract

A concept that has arisen over the last decade is that proteins can, in general, be covalently modified by polypeptides, resulting in alterations in their fate and function. The first?identified and most well studied of these modifying polypeptides is ubiquitin. Although targeting for proteasomal degradation is the best studied outcome of ubiquitylation, we now understand that modification of proteins with ubiquitin has numerous other cellular roles that alter protein function and that are unrelated to proteasomal degradation. Ubiquitylation is a complex process that is regulated at the level of both addition and removal of ubiquitin from target proteins. This unit includes a number of different basic protocols that will facilitate the study of components of the ubiquitin system and substrate ubiquitylation both in vitro and in cells. Because another protein modifier, NEDD8, itself regulates aspects of the ubiquitin system, basic protocols on neddylation are also included in this unit.

Keywords: Ubiquitin; ubiquitylation; proteasomal degradation; protein modification; NEDD8; E1; E2; E3

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Assays of E1 and E2 Activity
Basic Protocol 1: Thiolester Formation Between Rabbit E1 and Ubiquitin
Basic Protocol 2: Thiolester Formation Between E2 and Ubiquitin
Binding of Ubiquitin‐Proteasome Proteins
Basic Protocol 3: Binding of E2s to E3s
Alternate Protocol 1: Binding of Ubiquitin Substrates to E3s
In Vitro Ubiquitylation (E3) Assays
Basic Protocol 4: E3 Auto‐Ubiquitylation
Basic Protocol 5: Determination of Auto‐Ubiquitylation versus Pseudo‐Substrate Ubiquitylation
Basic Protocol 6: Ubiquitylation of E3 Enzymes Expressed in In Vitro Translation Systems
Alternate Protocol 2: Determination of Ubiquitin Chain Variant Phenotype
Basic Protocol 7: Chelation of Zinc from RING‐ and PHD‐Finger E3s
Alternate Protocol 3: Inhibition of HECT Domain E3s by Alkylation of Active‐Site Cysteines
Substrate Ubiquitylation in Vitro
Basic Protocol 8: Substrate Ubiquitylation in Solution
Alternate Protocol 4: Substrate Ubiquitylation After E3 Binding
Basic Protocol 9: Substrate Ubiquitylation by Multisubunit E3s
Basic Protocol 10: Detection of E3 Activity in Immunoprecipitated Protein
Alternate Protocol 5: Detection of Ubiquitin Modification of Substrates After GST Pull‐Down
Detection of Ubiquitylation in Vivo
Basic Protocol 11: Lysis and Immunoprecipitation of Ubiquitylated Protein
Alternate Protocol 6: Detection of Protein Ubiquitylation Using Tagged Ubiquitin
Inhibition of Ubiquitylation and Protein Degradation In Vivo
Basic Protocol 12: Use of Dominant Negative Ubiquitin Proteins in Cells
Alternate Protocol 7: Use of Small Interfering RNAs (siRNAs) to Reduce Expression of Ubiquitin‐Proteasome System Proteins in Cells
Basic Protocol 13: Localizing Degradation to the Proteasome
The NEDD8 Conjugation System
Basic Protocol 14: Purification of APP‐BP1/Uba3 Complex (E1 for NEDD8) Expressed Using the Baculovirus Expression System
Support Protocol 1: Batch Purification of APP‐BP1 and Uba3
Support Protocol 2: Column Purification of APP‐BP1 and Uba3
Basic Protocol 15: Production of Ubc12 and NEDD8 in Bacterial Expression Systems
Basic Protocol 16: In Vitro Neddylation
Labeling and Detection of Ubiquitin
Basic Protocol 17: Radiolabeling Ubiquitin with 32P
Alternate Protocol 8: Radiolabeling Ubiquitin with 125Iodine
Alternate Protocol 9: Labeling Ubiquitin with Nonradioactive Tag (Biotin)
Alternate Protocol 10: Generation of Tagged Ubiquitin Expression Plasmids
Basic Protocol 18: Immunoblotting with Anti‐Ubiquitin
Generation and Purification of Rabbit Anti‐Ubiquitin Antibodies
Basic Protocol 19: Generation and Purification of Anti‐Ubiquitin Antibodies
Support Protocol 3: Preparation of Ubiquitin Affinity Column
Generation of Reagent‐Grade Ubiquitin Activating Enzyme (E1)
Basic Protocol 20: Preparation of Wheat E1 in E. coli
Basic Protocol 21: Preparation of Mouse E1 in Insect Cells Using a Baculovirus System
Alternate Protocol 11: Preparation of Rabbit E1
Support Protocol 4: Generation of Empty Bacterial Lysates
Generation of Ubiquitin‐Conjugating Enzymes (E2s)
Basic Protocol 22: Expression of E2s in E. coli Without a Purification Tag
Alternate Protocol 12: Purification of E2s from E. coli Using an Affinity Tag
Alternate Protocol 13: Purification of E2s from E. coli Using Anti–Affinity Tag Antibodies
Basic Protocol 23: Expression of E2s with in Vitro Transcription and Translation Systems
In Vitro Production of Ubiquitin‐Protein Ligases (E3s)
Basic Protocol 24: Expression of Ring‐Finger E3s in Bacterial Lysates
Alternate Protocol 14: Expression of HECT and U‐Box E3s in Bacterial Lysates
Alternate Protocol 15: Expression of Unstable E3s in Bacterial Lysates
Basic Protocol 25: Expression of E3s in Eukaryotic in Vitro Translation Systems
Basic Protocol 26: Expression of Multi‐Subunit E3s Using a Baculovirus Expression System
Support Protocol 5: Batch Purification of Multimeric E3s
Support Protocol 6: Column Purification of Multimeric E3s
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Materials

Basic Protocol 1: Thiolester Formation Between Rabbit E1 and Ubiquitin Materials

E1: rabbit E1 (Calbiochem, Boston Biochem, Affiniti), bacterially expressed wheat E1 ( protocol 33), or mouse E1 ( protocol 34)
100 mM HEPES, pH 7.5
10× thiolester buffer (see recipe)
[32P]ubiquitin ( protocol 26) or 10 µg/µl “cold” (unlabeled) ubiquitin (Sigma) for immunoblot detection
4× nonreducing SDS‐PAGE sample buffer (see recipe)
4× reducing SDS‐PAGE sample buffer (see recipe)
6% to 8% SDS‐PAGE minigels (unit 6.1)
Anti‐ubiquitin antibody ( protocol 31; for immunoblot detection)
Boiling water bath

Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3) or immunoblotting ( protocol 30)

Basic Protocol 2: Thiolester Formation Between E2 and Ubiquitin Materials

E1: rabbit E1 (Calbiochem, Boston Biochem, Affiniti,), bacterially expressed wheat E1 ( protocol 33), or mouse E1 ( protocol 34)
100 mM HEPES, pH 7.5
E2: crude (Basic Protocol protocol 3722 or protocol 4023) or purified ( protocol 38 or protocol 3913) or commercially available
10× thiolester buffer (see recipe)
[32P]ubiquitin ( protocol 26)
4× nonreducing SDS‐PAGE sample buffer (see recipe)
4× reducing SDS‐PAGE sample buffer (see recipe)
18% to 20% SDS‐PAGE minigels (unit 6.1)
Anti‐ubiquitin antibody ( protocol 31; optional)
Boiling water bath

Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3) or immunoblotting ( protocol 30)

Basic Protocol 3: Binding of E2s to E3s Materials

E3 or other putative E2 binding site of interest expressed as a GST fusion protein (see protocol 41 and Alternate Protocols protocol 4214 and protocol 4315)
Glutathione‐Sepharose 4B beads (50% slurry; GE Healthcare)
Phosphate‐buffered saline (PBS; see recipe), 4°C
Binding buffer (see recipe), 4°C
Purified native E2 (see Alternate Protocols protocol 3812 and protocol 3913; for detection by Coomassie blue staining or immunoblotting), E2 fusion with GST, His or other tag (Wu et al., ; for detection by Coomassie blue staining or immunoblotting), or35S‐labeled E2 (see protocol 40; for detection by autoradiography)
4× nonreducing SDS‐PAGE sample buffer (see recipe)
4× reducing SDS‐PAGE sample buffer (see recipe)
18% to 20% SDS‐PAGE minigels (unit 6.1)
10% (v/v) acetic acid/25% methanol
Anti‐E2 or anti‐tag antibody (Calbiochem, Boston Biochem, Affinity; for immunoblotting)

End‐over‐end rotator
Boiling water bath
Gel densitometer

Additional reagents and equipment for Coomassie blue staining (unit 6.6), autoradiography and phospher imaging (unit 6.3), or immunoblotting (unit 6.2)

Alternate Protocol 1: Binding of Ubiquitin Substrates to E3s

In vitro–translated 35S‐labeled substrate protein ( protocol 44)
50 mM MG132 (Calbiochem, Boston Biochem, or Biomol) in DMSO
10% (v/v) acetic acid/25% (v/v) methanol

Basic Protocol 4: E3 Auto‐Ubiquitylation Materials

Glutathione‐Sepharose (GS) beads (GE Healthcare)
50 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
Putative E3 expressed as a GST fusion in bacteria (GST‐E3; protocol 44)
Empty bacterial lysate ( protocol 36)
Wheat E1 ( protocol 33), mouse E1 ( protocol 34), or rabbit E1 (Calbiochem)
E2 ( protocol 37)
10× PCK (see recipe)
10× ubiquitylation buffer 1 (see recipe)
[32P]ubiquitin ( protocol 26)
50 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
4× reducing SDS‐PAGE sample buffer (see recipe)
10% (v/v) acetic acid/25% methanol

30°C Thermomixer (Eppendorf), or other heated shaker
Boiling water bath

Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

Basic Protocol 5: Determination of Auto‐Ubiquitylation versus Pseudo‐Substrate Ubiquitylation Materials

Putative E3 expressed as a GST fusion in bacteria ( protocol 44)
Glutathione‐Sepharose (GS) beads (GE Healthcare)
Empty bacterial lysate ( protocol 36)
Wheat E1 ( protocol 33), mouse E1 ( protocol 34), or rabbit E1 (Calbiochem)
E2 ( protocol 37)
10× PCK (see recipe)
10× ubiquitylation buffer 1 (see recipe)
[32P]ubiquitin ( protocol 26)
50 mM Tris⋅Cl, pH 7.5
20× thrombin stock solution (see recipe)
Phosphate‐buffered saline (PBS; see recipe)
Ubiquitylation buffer 2 for E3 assays (see recipe; optional)
Thermomixer, or other heated shaker

Additional reagents and equipment for in vitro ubiquitylation ( protocol 6), SDS‐PAGE (unit 6.1), and autoradiography (unit 6.3).

Basic Protocol 6: Ubiquitylation of E3 Enzymes Expressed in In Vitro Translation Systems Materials

50 mM MG132 (Calbiochem, Boston Biochem, or BioMol) in DMSO
30 µM ubiquitin aldehyde (Calbiochem, Boston Biochem, or BioMol) in 1 M HEPES, pH 8.0
50 ng/µl E1 (Basic Protocols protocol 3421 and protocol 3722)
50 ng/µl E2 (e.g., Ube2d2/UbcH5B; protocol 37)
10× PCK (see recipe)
10× ubiquitylation buffer 1 (see recipe) or 20× ubiquitylation buffer 2 for E3 assays (see recipe)
10 mg/ml ubiquitin (Sigma)
Suspected E3 protein, expressed with 35S label in reticulocyte or wheat germ lysates (25,000 to 100,000 cpm; protocol 40)
4× reducing SDS sample buffer (see recipe)
10% (v/v) acetic acid/25% methanol
30°C Thermomixer (Eppendorf), or other heated shaker

Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

Alternate Protocol 2: Determination of Ubiquitin Chain Variant Phenotype

20× ubiquitylation buffer 2 for E3 assays (see recipe)
Mutant ubiquitins: lysine‐less (K0), K48R, K63R, K48 only, K63 only (BioMol, Boston Biochem, or made in house)
Wild‐type ubiquitin as reference

Additional reagents and equipment for anti‐ubiquitin immunoblotting ( protocol 30)

Basic Protocol 7: Chelation of Zinc from RING‐ and PHD‐Finger E3s Materials

Putative E3 expressed as a GST fusion in bacteria ( protocol 41).
Glutathione‐Sepharose (GS) beads (GE Healthcare)
50 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
Zinc chelating agents (Sigma): 5 to 10 mM EDTA, 5 to 10 mM EGTA, 5 to 10 mM DTPA, 1 to 5 mM TPEN, or 1 to 5 mM o‐phenanthroline in 50 mM Tris⋅Cl, pH 7.5
5 mM ZnCl 2
Wheat E1 ( protocol 33), mouse E1 ( protocol 34), or rabbit E1 (Calbiochem)
E2 ( protocol 37)
Empty bacterial lysate ( protocol 36)
10× PCK (see recipe)
10× ubiquitylation buffer 1 (see recipe)
[32P]ubiquitin ( protocol 26)
4× reducing SDS‐PAGE sample buffer (see recipe)

End‐over‐end rotator
Gel‐loading pipet tips
30°C Thermomixer (Eppendorf), or other heated shaker

Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

Alternate Protocol 3: Inhibition of HECT Domain E3s by Alkylation of Active‐Site Cysteines

Alkylating agents: 10 mM iodoacetamide (IAA) or 5 mM N‐ethylmaleamide (NEM) in Tris⋅Cl, pH 7.5 (see appendix 2A for buffer)

Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3) or immunoblotting (unit 6.2)

Basic Protocol 8: Substrate Ubiquitylation in Solution Materials

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