Mitochondrial Translocases Part B

1st Edition, Volume 707 - November 5, 2024
Imprint: Academic Press
Editor: Nils Wiedemann
Language: English
Hardback ISBN:
9 7 8 - 0 - 4 4 3 - 3 1 4 7 0 - 4
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 3 1 4 7 1 - 1

Mitochondrial Translocases Part B series, highlights new advances in the field, with this new volume presenting interesting chapters. Each chapter is written by an intern… Read more

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Mitochondrial Translocases Part B series, highlights new advances in the field, with this new volume presenting interesting chapters. Each chapter is written by an internation.

Title of Book
Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Preface
Section 1: Monitoring specific mitochondrial translocation steps and pathways
Chapter One: TOM-TIM23 supercomplex formation
Abstract
1 Introduction
2 Stabilization of the TOM-TIM23 supercomplex
3 Isolation of mitochondria
4 Expression and purification of Jac1sfGFP
5 In organello import of Jac1sfGFP
6 Isolation of the TOM-TIM23 supercomplex
Acknowledgments
References
Chapter Two: Analysis of inner membrane lateral sorting at the presequence translocase
Abstract
1 Introduction
2 Experimental design using Mgm1p
3 Preparation of plasmids
4 Assessment of the membrane insertion
5 Quantification and calculation of membrane insertion efficiencies
6 Conclusions
Acknowledgments
References
Chapter Three: Monitoring α-helical membrane protein insertion into the outer mitochondrial membrane of yeast cells
Abstract
1 Introduction
2 Experimental procedure
3 Concluding remarks
Acknowledgments
References
Chapter Four: Monitoring alpha-helical membrane protein insertion into the outer mitochondrial membrane in mammalian cells
Abstract
1 Introduction
2 Tools to visualize outer membrane protein biogenesis in cells
3 In vitro insertion assay
4 Reconstitution of α-helical membrane protein insertion using MTCH2 proteoliposomes
5 Summary and conclusions
Acknowledgments
References
Chapter Five: Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins
Abstract
1 Introduction
2 Before you begin
3 Key resource table
4 Materials and equipment
5 Step-by-step method details
6 Expected outcomes
7 Quantification and statistical analysis
8 Advantages
9 Limitations
10 Optimization and troubleshooting
11 Safety consideration and standards
Acknowledgment
References
Chapter Six: Analysis of protein trafficking between mitochondria and the endoplasmic reticulum by fluorescence microscopy
Abstract
1 Introduction
2 Strategy to analyze protein transfer in living cells
3 Construction of yeast strains for live cell imaging
4 Time-lapse fluorescence microscopy
5 Summary and conclusion
Acknowledgments
References
Chapter Seven: Monitoring retro-translocation of proteins from the mitochondrial intermembrane space
Abstract
1 Introduction
2 The principle of retro-translocation assay
3 General considerations
4 Preparation of the mitochondrial sample. Yeast and human cell culture conditions and mitochondria isolation procedures
5 IMS protein retro-translocation assay
6 Monitoring proteolytic release of stalled import intermediates
7 Summary and conclusion
8 Key resources table
Acknowledgments
References
Chapter Eight: An introduction to comparative genomics, EukProt, and the reciprocal best hit (RBH) method for bench biologists: Ancestral phosphorylation of Tom22 in eukaryotes as a case study
Abstract
1 Introduction to homology searching
2 Problems with paralogy: Homologues, orthologues, paralogues
3 The reciprocal best hit method
4 Taxon sampling is important: EukProt to the rescue!
5 Opisthokonts and other important taxonomic nomenclature
6 Daisy-chaining the RBH method
7 Hidden Markov model searching
8 Building trees resolves paralogous gene families
9 Answering questions with downstream analyses
10 Introduction to phosphorylation of Tom22
11 Results with step-by-step methods
12 Three putative Tom22 phosphorylation regions are conserved in animals, fungi, and related protists
13 Most EukProt predicted proteomes contain a Tom22 with predicted phosphorylation sites
14 Conclusions
Acknowledgments
References
Section 2: Analysis of mitochondrial protein complexes
Chapter Nine: Analysis of protein-protein interaction of the mitochondrial translocase at work by using technically effective BPA photo-crosslinking method
Abstract
1 Introduction
2 In vivo pBpa introduction and photo-crosslinking
3 Sample preparation and analysis of in vivo photo-crosslinking products
4 Mitochondrial isolation
5 In organello mitochondrial preprotein photo-crosslinking purification and analysis
6 Conclusion
Acknowledgements
References
Chapter Ten: Analysis of mitochondrial protein translocation by disulfide bond formation and cysteine specific crosslinking
Abstract
1 Introduction
2 Experimental design and general considerations
3 Tris/Tricine-SDS-PAGE
4 Analysis of the subunit arrangement of mitochondrial translocases
5 Analysis of precursor protein translocation by oxidation or cysteine specific crosslinking
6 Analysis of oxidation or cysteine specific crosslinking experiments
7 Summary
Acknowledgments
References
Chapter Eleven: STED super-resolution microscopy of mitochondrial translocases
Abstract
1 Introduction
2 Immunofluorescence labeling and STED microscopy of TOM20 and DNA
Acknowledgments
References
Chapter Twelve: Analysis of mitochondrial translocases TOM and TIM by the patch-clamping technique
Abstract
1 Introduction
2 Mitochondria and mitochondrial membranes for electrophysiology
3 Mitochondrial patch-clamping
4 Insides of the patch-clamp technique to study mitochondrial protein import
5 Electrophysiological hallmarks of the TOM, TIM23, and TIM22 channels
6 Conclusions
Acknowledgments
References
Chapter Thirteen: Large-scale purification of mitochondrial protein complexes in yeast expression system for structural analyses
Abstract
1 Introduction
2 Yeast overexpression strain
3 Large-scale protein purification
4 Differences in sample preparation between cryo-EM and HS-AFM analyses
5 Expected outcomes and advantages
Acknowledgments
References
Section 3: Mitochondrial protein folding, assembly and aggregation
Chapter Fourteen: Generation of TIM chaperone substrate complexes
Abstract
1 Introduction
2 General considerations about preparing chaperone-client complexes
3 Production and purification of tim chaperones
4 Preparing chaperone—Client complex with a pull-down approach
5 Complex formation in a cell-free protein production system
6 Appendix
References
Chapter Fifteen: Purification of functional recombinant human mitochondrial Hsp60
Abstract
1 Introduction
2 Materials and equipment
3 Expression of human mitochondrial Hsp60 in E. coli
4 Purification of the protein
5 Cryo-EM for high-resolution structures of Hsp60 oligomers and complexes
6 Pitfalls
Acknowledgments
References
Chapter Sixteen: Generating mammalian knock-out cell lines to investigate mitochondrial protein complex assembly
Abstract
1 Introduction
2 Generation of CRISPR/Cas9 constructs
3 Transfection of CRISPR/Cas9 constructs and isolation of clonal cell populations
4 Screening and validation of successful gene-editing
5 Retroviral complementation
6 Basic functional characterization of an OXPHOS assembly defect using knockout cell lines
References
Chapter Seventeen: Analysis of mitochondrial protein aggregation and disaggregation
Abstract
1 Introduction
2 Detection and identification of aggregated mitochondrial polypeptides
3 Disaggregation of mitochondrial polypeptides
4 Aggregation control by sequestration
5 Conclusion and outlook
References
Section 4: Regulation of mitochondrial protein translocation
Chapter Eighteen: Monitoring protein phosphorylation at the mitochondrial protein import machinery by PhosTag electrophoresis
Abstract
1 Introduction
2 Monitoring protein phosphorylation in isolated mitochondria
3 Monitoring protein phosphorylation of import receptors in vitro
4 Summary
Acknowledgments
References
Chapter Nineteen: Analysis of mitochondrial biogenesis regulation by oxidative stress
Abstract
1 Introduction
2 Methods to identify oxidation
3 Summary
Acknowledgment
References
Section 5: Mitochondrial unfolded protein response and protein degradation
Chapter Twenty: Methods to analyze the mitochondrial unfolded protein response (UPRmt)
Abstract
1 Introduction
2 Measuring mitochondrial UPR activation
3 Imaging mitochondria via fluorescence microscopy
4 Mitochondrial fractionation
5 Detection of mitochondrial targeting sequence (MTS) cleavage upon import into the mitochondrial matrix
6 Analysis of respiration
7 mtDNA quantification
8 ChIP–mtDNA followed by mtDNA quantification
Acknowledgments
References
Chapter Twenty One: Analysis of quality control pathways for the translocase of the outer mitochondrial membrane
Abstract
1 Introduction
2 Methods
3 Affinity purification of ubiquitin-modified precursor proteins
4 Analysis of precursor turnover at the TOM complex
Acknowledgements
References
Chapter Twenty Two: Analysis of the mitochondria-associated degradation pathway (MAD) in yeast cells
Abstract
1 Introduction
2 Before you begin
3 Key resources table
4 Materials and equipment
5 Step-by-step method details
6 Expected outcomes
7 Advantages
8 Limitations
9 Optimization and troubleshooting
10 Conclusion/summary
Acknowledgments
References
Chapter Twenty Three: Screen for temperature-sensitive mutants of non-essential yeast genes
Abstract
1 Introduction and overview
2 Generation of a synthetic lethal strain
3 Generation of a mutant library
4 Transforming and screening mutant libraries
5 Validation of temperature-sensitive candidate alleles
6 Conclusion
Acknowledgments
References
Section 6: Mitochondrial integrity
Chapter Twenty Four: Approaches for the analysis of redox-dependent protein import into mitochondria of mammalian cells
Abstract
1 Introduction
2 Assessment of the cysteine redox state of mitochondrial proteins at steady state in intact cells (direct and inverse shift assay)
3 Following MIA40-substrate interaction
4 Following oxidation of mitochondrial proteins over time (oxidation kinetics)
5 Following translocation of proteins by import of radioactively labelled precursors into isolated mitochondria
6 Manipulation of the redox environment in the IMS to influence redox-dependent mitochondrial import
References
Chapter Twenty Five: Seahorse assay for the analysis of mitochondrial respiration using Saccharomyces cerevisiae as a model system
Abstract
1 Introduction
2 Experimental overview
3 Materials and equipment
4 Protocol
5 Examples
6 Limitations
7 Conclusion
Acknowledgments
References

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Mitochondrial Translocases Part B

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