Plant Proteostasis: Methods and Protocols

Preface ``Plant Proteostasis´´
Contents
Contributors
Part I: Ubiquitin Conjugation and Deconjugation Analysis
	Chapter 1: Observing Real-Time Ubiquitination in High Throughput with Fluorescence Polarization
		1 Introduction
		2 Materials
			2.1 Enzymes Required for the Ubiquitination Reaction
			2.2 Fluorescent Probes
			2.3 Protein Concentrations and Buffer Conditions
			2.4 Plate Reader and Assay Parameters
		3 Methods
			3.1 Monitoring Activity of E3 Ubiquitin Ligases
			3.2 Applying UbiCRest to UbiReal to Determine Poly-Ubiquitin Linkage Types
			3.3 UbiReal to Quantify Inhibitor Potency
			3.4 Data Analysis
		4 Notes
		References
	Chapter 2: Identification and Characterization of Physiological Pairing of E2 Ubiquitin-Conjugating Enzymes and E3 Ubiquitin L...
		1 Introduction
		2 Materials
			2.1 BiFC: Plant Growth for Protoplast Isolation
			2.2 BiFC: Plasmids and Bacteria
			2.3 BiFC: Protoplast Isolation and Transformation
				2.3.1 Solutions and Buffer
				2.3.2 Enzymes
			2.4 Autoubiquitination: Plasmids and Bacteria
			2.5 Autoubiquitination: Protein Expression and Purification
			2.6 Autoubiquitination: SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and  WB
			2.7 Autoubiquitination: Antibodies
			2.8 Laboratory Equipment
		3 Methods
			3.1 BiFC: Protoplast Transformation
			3.2 Autoubiquitination: E2-E3 Pair Activity Assay
				3.2.1 Autoubiquitination: Reconstitution of Ubiquitination in Bacteria Using Determined E2-E3 Pairs
				3.2.2 Autoubiquitination: Purification of Ubiquitinated Products
			3.3 Autoubiquitination: SDS-PAGE and WB Analysis
		4 Notes
		References
	Chapter 3: Immunoprecipitation of Cullin-Ring Ligases (CRLs) in Arabidopsis thaliana Seedlings
		1 Introduction
		2 Materials
			2.1 Co-IP of  CRLs
				2.1.1 Plant Material and Growth
				2.1.2 Total Protein Extraction
				2.1.3 Immunoprecipitation
				2.1.4 SDS-PAGE
				2.1.5 Immunoblotting and Detection
			2.2 In Vivo Ubiquitination Analysis of  CRLs
				2.2.1 Plant Material and Growth
				2.2.2 Total Protein Extraction
				2.2.3 Immunoprecipitation
				2.2.4 SDS-Page
				2.2.5 Immunoblot and Detection
		3 Methods
			3.1 IP of  CRLs
				3.1.1 Plant Growth Conditions and Cross-Link
				3.1.2 Total Protein Extraction
				3.1.3 Immunoprecipitation
				3.1.4 SDS-Page
				3.1.5 Immunoblot and Detection
			3.2 In Vivo Ubiquitination Analysis of  CRLs
				3.2.1 Plant Material and Growth
				3.2.2 Total Protein Extraction
				3.2.3 Immunoprecipitation
				3.2.4 SDS-PAGE
				3.2.5 Immunoblot and Detection
		4 Notes
		References
	Chapter 4: An In vitro Assay to Recapitulate Hormone-Triggered and SCF-Mediated Protein Ubiquitylation
		1 Introduction
		2 Materials
			2.1 In Vitro Ubiquitylation (IVU) Protein Components (See Note 1)
			2.2 In Vitro Ubiquitylation (IVU) Reagents
			2.3 Quantification and Evaluation of Ubiquitylation
		3 Methods
			3.1 In Vitro Reconstitution of the Ubiquitylation Cascade
			3.2 Detection of Ubiquitylated Substrates
			3.3 Quantification of Ubiquitylated Substrates and Data Analysis
		4 Notes
		References
	Chapter 5: Analysis of Proteasome-Associated Ubiquitin Ligase Activity
		1 Introduction
		2 Materials
			2.1 Plant Growth and Sample Collection
			2.2 Laboratory Equipment
			2.3 Immunopurification of the Proteasome and Proteasome-Interacting Proteins
			2.4 Ubiquitination Assay
			2.5 Purification of Ubiquitin Conjugates
			2.6 Western Blotting
		3 Methods
			3.1 Plant Growth and Sample Collection
			3.2 Immunopurification of the Proteasome and Proteasome-Interacting Proteins
			3.3 Ubiquitination Assay
			3.4 Purification of Free Ubiquitin Conjugates
				3.4.1 Expression and Purification of Proteins in Bacteria
				3.4.2 Purification of Free Ubiquitin Conjugates
			3.5 PAGE and Western Blotting
		4 Notes
		References
	Chapter 6: Measuring the DUB Activity of Arabidopsis Deubiquitylating Enzymes
		1 Introduction
		2 Materials
			2.1 Recombinant GST-Tagged DUB Purification from Bacteria
			2.2 Deubiquitylation Assay (DUB Assay)
			2.3 Gradient Gel Electrophoresis
			2.4 Protein Transfer and Western Blotting
			2.5 Coomassie Brilliant Blue (CBB) Staining
			2.6 Fluorescence-Based DUB Assay
		3 Methods
			3.1 Recombinant DUB Purification from Bacteria
			3.2 Deubiquitylation Assay
			3.3 Gel Electrophoresis
			3.4 Gel Transfer and Western Blotting
			3.5 Coomassie Brilliant Blue (CBB) Staining
			3.6 DUB Assay Using a Fluorescent Substrate
		4 Notes
		References
Part II: Sumo Conjugation and Deconjugation
	Chapter 7: SUMO Conjugation and SUMO Chain Formation by Plant Enzymes
		1 Introduction
		2 Materials
			2.1 Components of the SUMOylation System
			2.2 Expression of Recombinant Plant SUMOylation Enzymes in E. coli
			2.3 Purification of His-Tagged Proteins (See Notes 2 and 3)
			2.4 Purification of MBP-Tagged Proteins
			2.5 Purification of Untagged SCE1
			2.6 FPLC Purification
			2.7 In Vitro SUMO Conjugation to Substrates and SUMO Chain Formation
			2.8 Detection of SUMO Conjugates on Western blots
		3 Methods
			3.1 Expression of Recombinant Plant SUMOylation Enzymes in E. coli
			3.2 Purifying His-Tagged Proteins (SUMO and SAE)
			3.3 Purification of MBP-Tagged Proteins (PIAL1 and PIAL2)
			3.4 Purification of Untagged SCE1
			3.5 In vitro SUMOylation Assay (Fig. 1)
			3.6 In Vitro SUMO Chain Formation. Small Scale Reaction for Western blot Detection (Fig. 2)
			3.7 In Vitro SUMO Chain Formation. Large-Scale Reaction for Isolation of SUMO Chains
			3.8 SUMO Protease Activity Test Using SUMO Chains (Fig. 3)
		4 Notes
		References
	Chapter 8: Kinetic Analysis of Plant SUMO Conjugation Machinery
		1 Introduction
		2 Materials
			2.1 Expression and Purification of Enzymes
			2.2 SUMO Conjugation Assays
		3 Methods
			3.1 Preparation of Recombinant SUMO Machinery Components: Expression and Purification of SUMO Isoforms and the SUMO-Conjugatin...
			3.2 Preparation of Recombinant SUMO Machinery Components: Expression and Purification of E1-Activating Enzyme Isoforms (SAE2/S...
			3.3 Preparation of Recombinant SUMO Conjugation Substrate: Expression and Purification of GST-CAT3Ct
			3.4 In vitro SUMOylation Assays for Analyzing Distinct SUMO Isoforms
			3.5 Quantification of SUMO Conjugation Kinetics
		4 Notes
		References
	Chapter 9: Expression, Purification, and Enzymatic Analysis of Plant SUMO Proteases
		1 Introduction
		2 Materials
			2.1 Bacterial Culture and Purification of Arabidopsis AtOTS1 SUMO Protease and HisSUMO1-FLC Recombinant Proteins
			2.2 SUMO Protease Assay
		3 Methods
			3.1 Expression of Recombinant SUMO Protease Protein
			3.2 Purification of Bacterially Expressed OTS1 Protein Using Small-Scale Batch Method
			3.3 Purification of Covalently Conjugated 6XHIS-SUMO1-FLC: SUMO Protease Substrate from Bacterial Expression System Using Smal...
			3.4 SUMO Protease Activity Assays Using a SUMO-Linked Substrate
		4 Notes
		References
Part III: Analysis of Autophagy
	Chapter 10: Monitoring Autophagic Flux in the Model Single-Celled Microalga Chlamydomonas reinhardtii
		1 Introduction
		2 Materials
			2.1 Growth Media and Components
				2.1.1 Tris-Acetate Phosphate (TAP) Medium
				2.1.2 High-Salt Medium (HSM)
			2.2 Drugs for Blocking Autophagic Flux
			2.3 Drugs for Activating Autophagic Flux
			2.4 SDS-PAGE Components
			2.5 Protein Preparation Components
			2.6 Immunoblot Components
		3 Methods
			3.1 Preparation of Soluble Protein Extracts from Chlamydomonas
			3.2 Separation and Analysis of Proteins by Electrophoresis
			3.3 Western Blotting and ATG8 Protein Detection
		4 Notes
		References
	Chapter 11: Detection of Autophagy in Plants by Fluorescence Microscopy
		1 Introduction
		2 Materials
			2.1 Plant Growth Materials
			2.2 Seedling Treatment and Staining
			2.3 Fluorescence Microscopy
		3 Methods
			3.1 Plant Materials and Growth Conditions
			3.2 Autophagy Activation in Seedlings by Abiotic Stresses (See Note 7)
			3.3 Labeling of Autophagosomes in Seedlings by MDC Staining
			3.4 Labeling of Autophagosomes in Seedlings with GFP-ATG8 Fusion Protein (See Note 10)
			3.5 Visualization of MDC-Stained or GFP-Labeled Autophagosomes by Fluorescence Microscopy
		4 Notes
		References
	Chapter 12: Characterization of ATG8-Family Interactors by Isothermal Titration Calorimetry
		1 Introduction
			1.1 Structure and Function of ATG8 in Autophagy
			1.2 Models and Methods to Characterize ATG8 Interactors
			1.3 ITC of Heterodimeric Interactions
			1.4 ITC Data Analysis
		2 Materials
			2.1 ATG8A Expression
			2.2 ATG8A Purification
			2.3 ITC Assays
			2.4 Data Analysis and Presentation
		3 Methods
			3.1 AtATG8A Protein Expression
			3.2 AtATG8A Purification
			3.3 Peptide Preparation
			3.4 Water-Water Titrations
			3.5 Chemical Test Reaction
			3.6 Direct Ligand Titration Assay
			3.7 Displacement Assay
			3.8 Alternative Competition Assay
			3.9 Software Setup
			3.10 Thermogram Integration
			3.11 Global Analysis in SEDPHAT for the Direct Ligand Titration Experiments
				3.11.1 Displacement Assay: Simplified Case Scenario
			3.12 Global Analysis in SEDPHAT for the Displacement Assays Using the Available sAIM Peptide Direct Titrations (Assuming the D...
			3.13 Global Analysis in SEDPHAT for the Displacement Assays Using the Available AIM Peptide Direct Titrations (Assuming the sA...
			3.14 Global Analysis in SEDPHAT for the Alternative Competition Assay Using the AIM as Known Competitor
			3.15 Data Presentation
			3.16 Data Interpretation and Conclusions
		4 Notes
		References
Part IV: Protein Turnover and Stability
	Chapter 13: Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System
		1 Introduction
		2 Materials
			2.1 Maintenance of Arabidopsis Cell Suspension Culture, Preparation of Arabidopsis Protoplasts, and Transfections
			2.2 Metabolic Labeling and Pulse-Chase Experiments
			2.3 Stability Measurements Using Reporter-Dependent Assays
		3 Methods
			3.1 Preparation of Arabidopsis Protoplasts and Transfection
			3.2 Pulse-Chase Experiments Using Radioactive Metabolic Labeling of Transfected Protoplasts
			3.3 Stability Measurements Using Reporter-Dependent Assays
		4 Notes
		References
	Chapter 14: Relative Protein Lifetime Measurement in Plants Using Tandem Fluorescent Protein Timers
		1 Introduction
		2 Materials
			2.1 Agrobacterium and Plant Transformation
			2.2 Protein Extraction
			2.3 Immunoblotting
			2.4 Microscopy
			2.5 Selection and Treatment of Transgenic Arabidopsis
		3 Methods
			3.1 Preparation of Electrocompetent Agrobacterium tumefaciens Cells
			3.2 Transformation and Culture of Agrobacterium tumefaciens
			3.3 Transient Transformation of Nicotiana benthamiana by Agroinfiltration
			3.4 Protein Extraction and Immunoblotting
			3.5 Confocal Microscopy of Transiently Transfected N. benthamiana
			3.6 Confocal Microscopy of Arabidopsis
			3.7 Strategy for Selection of Arabidopsis thaliana Stably Expressing tFTs
			3.8 Proteasome Inhibitor Treatments
		4 Notes
		References
	Chapter 15: Detection and Quantification of Protein Aggregates in Plants
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Confocal Microscopy
			2.3 Aggregate Protein Isolation
		3 Methods
			3.1 Plant Growth Conditions and Stress Treatment
			3.2 Extraction of Protein Aggregates
			3.3 Confocal Microscopy
			3.4 Volocity 3D Image Analysis Software to Quantify Aggregates
			3.5 Analysis of Aggregates and Statistical Analysis (Scheme of the Process Shown in Fig. 1)
		4 Notes
		References
	Chapter 16: Using Intrinsic Fluorescence to Measure Protein Stability Upon Thermal and Chemical Denaturation
		1 Introduction
		2 Materials
			2.1 pH Denaturation
			2.2 Thermal Denaturation
			2.3 Chemical Denaturants
		3 Methods
			3.1 Protein Stock Solution
			3.2 Incubation of Proteins into Different pH Solutions
			3.3 Preparation of Titration Curves Ranging 0-8 M Urea
			3.4 Preparation of Titration Curves Ranging 0-6 M Guanidine Hydrochloride
			3.5 Preparation of Melting Point Curves
			3.6 Measurements of pH Stability Using Spectrofluorometer
			3.7 Measurements of Titration Curves Using Spectrofluorometer
			3.8 Recording Melting Point Curves Using Spectrofluorometer
			3.9 Data Plotting and Thermodynamic Parameters
		4 Notes
		References
Part V: Protein Isolation and Mass Spectrometric Analysis
	Chapter 17: Purification and Detection of Ubiquitinated Plant Proteins Using Tandem Ubiquitin Binding Entities
		1 Introduction
		2 Materials
			2.1 Plant Growth Conditions
			2.2 Expression of Target Protein: Agrobacterium-Mediated Transient Expression
			2.3 TUBE Assay
			2.4 Detection and Analysis
			2.5 Equipment
		3 Methods
			3.1 Plant Growth Conditions
			3.2 Expression of the Target Protein: Agrobacterium-Mediated Transient Expression
			3.3 Sample Preparation and TUBE Assay
			3.4 Analysis of Ubiquitination by Immunoblotting
		4 Notes
		References
	Chapter 18: Titanium Oxide-Based Phosphopeptide Enrichment from Arabidopsis Seedlings
		1 Introduction
		2 Materials
			2.1 Seedling Growth
			2.2 Protein Extraction and Digestion
				2.2.1 C18 Solid Phase Extraction (SPE)
				2.2.2 TiO2 Enrichment
				2.2.3 Microspin C18 SPE Cleanup
		3 Methods
			3.1 Growing Seedlings and Harvesting Tissue
			3.2 Protein Extraction
			3.3 In-Solution digestion
			3.4 In-Solution Digestion Cleanup
			3.5 TiO2 Enrichment
			3.6 Microscale SPE Cleanup
		4 Notes
		References
	Chapter 19: Chloroplast Envelope Membrane Subfractionation from Arabidopsis and Pea
		1 Introduction
		2 Materials
			2.1 Plant Growth
			2.2 Chloroplast Isolation and Envelope Membrane Subfractionation of Arabidopsis
			2.3 Chloroplast Isolation and Envelope Membrane Subfractionation of Pea
			2.4 SDS-PAGE
			2.5 Semidry Western Blot
			2.6 Standard In Vitro Transcription and Translation
			2.7 In Vitro Transcription and Translation with the TNT-Coupled Reticulocyte Lysate System by Promega
			2.8 In Vitro Import and Subsequent Sublocalization
		3 Methods
			3.1 Plant Growth
			3.2 Chloroplast Isolation of Arabidopsis and Pea
				3.2.1 Chloroplast Isolation from Arabidopsis
				3.2.2 Chloroplast Isolation from Pea
			3.3 Chloroplast Envelope Membrane Subfractionation
				3.3.1 Chloroplast Envelope Membrane Subfractionation from Arabidopsis
				3.3.2 Chloroplast Envelope Membrane Subfractionation from Pea
			3.4 SDS-PAGE
			3.5 Semidry Western Blotting
			3.6 Dynamic Sublocalization During Plant Development
			3.7 In Vitro Transcription and Translation
			3.8 In Vitro Import and Subsequent Sublocalization
		4 Notes
		References
	Chapter 20: Chromatin Enrichment for Proteomics in Plants (ChEP-P)
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Preparation of Chromatin Enrichment for Proteomics (ChEP)
			2.3 Trypsin Digestion
			2.4 MS/MS Analysis
			2.5 Data Processing
		3 Methods
			3.1 Chromatin Cross-Linking
			3.2 Lysis
			3.3 Chromatin Enrichment
			3.4 Gel Analysis
			3.5 In-solution Trypsin Digestion
			3.6 Nano-HPLC-MS/MS Analysis
			3.7 Database Search and Quantitation
			3.8 Data Processing and Statistical Analysis
		4 Notes
		References
	Chapter 21: Proximity-Dependent In Vivo Biotin Labeling for Interactome Mapping in Marchantia polymorpha
		1 Introduction
		2 Materials
			2.1 Plant Growth
			2.2 Biotin Treatment and Protein Extraction
			2.3 Biotin Depletion
			2.4 Affinity Pull-Down
			2.5 On-bead Digestion
		3 Methods
			3.1 Plant Growth
			3.2 Biotin Treatment
			3.3 Total Protein Extraction
			3.4 Biotin Depletion by MeOH:CHCl3 Precipitation
			3.5 Affinity Pull-Down of Biotinylated Proteins
			3.6 On-bead Digestion with Trypsin
		4 Notes
		References
	Chapter 22: Tandem Mass Tag-Based Phosphoproteomics in Plants
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Preparation of Protein Extracts
			2.3 Protein Precipitation
			2.4 Trypsin Digestion
			2.5 TMT Labeling
			2.6 Phosphopeptide Enrichment and High pH Reversed-Phase Fractionation
			2.7 MS/MS Analysis
			2.8 Protein Identification
		3 Methods
			3.1 Sample Preparation
			3.2 Protein Precipitation
			3.3 In-Solution Trypsin Digestion
			3.4 TMT Labeling
			3.5 Phosphopeptide Enrichment and High pH Reversed-Phase Fractionation
			3.6 Nano-HPLC-MS/MS Analysis
			3.7 Database Search
			3.8 Data Processing and Statistical Analysis
		4 Notes
		References
Part VI: Study of Proteasome and Proteases
	Chapter 23: Analysis of Peptide Hormone Maturation and Processing Specificity Using Isotope-Labeled Peptides
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Synthetic Peptides
			2.3 Plant Material, Constructs, Bacteria, and Media
			2.4 Transient Expression in N. benthamiana and Purification of SBT5.4
			2.5 Sample Preparation for MS Analysis
			2.6 MS Analysis
		3 Methods
			3.1 Digestion of the CIF4 Precursor Peptide and Its Sulfated Isotopolog by SBT5.4
				3.1.1 Transient Expression of SBT5.4 by Agro-infiltration of N. benthamiana Leaves
				3.1.2 Purification of Recombinant SBT5.4
				3.1.3 Peptide Digest
			3.2 Analysis of Cleavage Products by MS
				3.2.1 Sample Preparation (Desalting) for MS Analysis (See Note 8)
				3.2.2 Nano-LC-ESI-MS/MS
				3.2.3 MS Data Analysis
		4 Notes
		References
	Chapter 24: Improved Identification of Protease Cleavage Sites by In-gel Reductive Dimethylation
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 SDS-PAGE
			2.3 In-gel Reductive Dimethylation and Tryptic Digest
			2.4 MS Analysis
		3 Methods
			3.1 SDS-PAGE Analysis of Cleavage Products
			3.2 In-gel Reductive Dimethylation and Tryptic Digest
			3.3 Nano-LC-ESI-MS/MS Analysis
		4 Notes
		References
	Chapter 25: A Pipeline to Monitor Proteasome Homeostasis in Plants
		1 Introduction
		2 Materials
			2.1 Proteasome Activity Measurement with Luminogenic Proteasome Substrate
			2.2 Evaluation of Total Ubiquitinated Proteins
			2.3 Monitoring of Proteasome Stress Regulon mRNA Levels
			2.4 Evaluation of Proteasome Subunit Protein Level
		3 Methods
			3.1 Characterization of Proteasome Activity in Plants
				3.1.1 Proteasome Activity Measurement with Luminogenic Proteasome Substrate
				3.1.2 Evaluation of Total Ubiquitinated Proteins
			3.2 Characterization of Proteasome Transcript Regulation and Protein Abundance
				3.2.1 Monitoring of Proteasome Stress Regulon mRNA Levels
				3.2.2 Evaluation of Proteasome Subunit Protein Level
		4 Notes
		References
Part VII: Bioinformatic Analysis
	Chapter 26: Bioinformatic Tools for Exploring the SUMO Gene Network: An Update
		1 Introduction
		2 Materials
		3 Methods
			3.1 The SUMO Gene Network
				3.1.1 Interpreting SGN Datasets
				3.1.2 Managing SGN Datasets
			3.2 In Silico Prediction of SUMO Attachment Sites and SIMs
			3.3 Comparative Genomics for Ortholog Identification
			3.4 Functional Categorization and Gene Network Analysis
		4 Notes
		References
	Chapter 27: Coexpression Network Construction and Visualization from Transcriptomes Underlying ER Stress Responses
		1 Introduction
			1.1 WGCNA
			1.2 Cytoscape
		2 Materials
		3 Methods
			3.1 Preparation
			3.2 Coexpression Network Analysis Using WGCNA
				3.2.1 Data Loading and Cleaning
				3.2.2 Choosing the Soft-Threshold Power to Fit a Scale-Free Topology to the Network
				3.2.3 Identifying Coexpression Similarity and Adjacency
				3.2.4 Module Detection
				3.2.5 Merging Modules with Similar Expression Profiles
				3.2.6 Visualization of Weighted Networks and Exporting Modules
				3.2.7 Calculating and Visualizing Module Eigengenes
				3.2.8 Extracting Expression Profiles of Eigengenes and Module Memberships
			3.3 Network Visualization Using Cytoscape
		4 Notes
		References
	Chapter 28: Approaches for the Identification of Intrinsically Disordered Protein Domains
		1 Introduction
		2 Materials
		3 Methods
			3.1 Identification of Protein Domains
			3.2 Prediction of Protein Disorder
			3.3 Identification of IDD
		4 Notes
		References
Index