Mitochondrial Translocases Part A

1st Edition, Volume 706 - October 24, 2024
Imprint: Academic Press
Editor: Nils Wiedemann
Language: English
Hardback ISBN:
9 7 8 - 0 - 4 4 3 - 2 9 3 3 0 - 6
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 2 9 3 3 1 - 3

Mitochondrial Translocases Part A 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 A series, highlights new advances in the field, with this new volume presenting interesting chapters. Each chapter is written by an international board of authors.

Title of Book
Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Preface
Section 1 Isolation of mitochondria
Chapter One: Isolation of yeast mitochondria by differential centrifugation
Abstract
1 Introduction
2 Before you begin
3 Key resources table
4 Materials and equipment
5 Step-by-step method details
6 Expected outcomes for isolated yeast mitochondria
7 Advantages
8 Limitations
9 Optimization and troubleshooting
10 Safety considerations and standards
Acknowledgments
References
Chapter Two: Isolation of yeast mitochondria by affinity purification using magnetic beads
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 Alternative methods/procedures
11 Summary/Conclusion
Acknowledgments
References
Chapter Three: A modified procedure for separating yeast peroxisomes from mitochondria
Abstract
1 Introduction
2 Yeast growth conditions
3 Preparation of yeast spheroplasts
4 Preparation of yeast cell lysates and isolation of crude organelles
5 Discontinuous gradient to separate mitochondria and peroxisomes
Acknowledgments
References
Section 2 Analysis of mitochondrial proteins
Chapter Four: Subcellular fractionation by differential centrifugation for mitochondrial studies
Abstract
1 Introduction
2 General considerations
3 Experimental design
4 Subcellular fractionation of yeast cells by differential centrifugation
5 Conclusion and summary
Acknowledgments
References
Chapter Five: Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP
Abstract
1 Introduction
2 Materials
3 Methods
Acknowledgments
References
Chapter Six: Analysis of mitochondrial biogenesis and protein localization by genetic screens and automated imaging
Abstract
1 Introduction to yeast genetics
2 Systematic mutant collections: past and present
3 Creation of “tailor made” collections
4 Interrogating collections
5 Discovery of mitochondrial biology using systematic mutant libraries
6 Perspective
Acknowledgments
References
Chapter Seven: Methods to monitor mitochondrial disulfide bonds
Abstract
Abbreviations
1 Introduction
2 Strategies to monitor mitochondrial disulfide bonds
3 Direct and indirect thiol trap assays
4 Strategies to identify MIA40 substrates
5 Import of Mia40 substrates
Acknowledgment
References
Section 3 Prediction, analysis and processing of mitochondrial targeting signals
Chapter Eight: Prediction of mitochondrial targeting signals and their cleavage sites
Abstract
1 Introduction
2 Use and installation of MTS prediction tools
3 Resources for MTS dataset construction
4 Retrospective case study
5 Summary of computational methods used for MTS prediction
6 Regarding predictions on “atypical” proteins
7 Future directions of MTS and similar prediction tasks
8 Summary
Acknowledgments
References
Chapter Nine: Monitoring mitochondrial precursor processing and presequence peptide degradation
Abstract
1 Introduction
2 Analysis of MPP activity in vitro
3 Analysis of precursor processing and presequence degradation in mitochondrial soluble extracts
Acknowledgments
References
Chapter Ten: Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry
Abstract
1 Introduction
2 Enrichment of N-terminal peptides
3 N-terminome data analysis and termini annotation
4 Expected outcomes
5 Alternative methods
Acknowledgments
References
Chapter Eleven: Analysis of targeting signals for mitochondrial intermembrane space import
Abstract
Abbreviations
1 Introduction
2 Precursors and import assays
3 Post import analysis and sub-fractionation
4 Overview and conclusions
Acknowledgments
References
Chapter Twelve: Analysis and prediction of internal mitochondrial targeting signals
Abstract
1 Introduction
2 Mitochondrial protein import: Routes and targeting sequences
3 Internal targeting sequences: The challenge of iMTS identification
4 Machine learning as a generic approach for iMTS identification
5 iMTS prediction with TargetP
6 Facilitating iMTS identification using the predictor iMLP
7 Limitations
8 Optimization and troubleshooting
9 Concluding remark
Acknowledgments
References
Section 4 Mitochondrial precursor proteins in the cytosol
Chapter Thirteen: Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol
Abstract
1 Introduction
2 Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol of yeast
3 Detection of mitochondrial precursor protein aggregates via immunofluorescence microscopy
4 Studying the effects of mitochondrial precursor aggregation on cellular proteostasis via fluorescence microscopy
5 Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol of human cells
6 Overview and future perspectives
Acknowledgments
References
Chapter Fourteen: In vitro translation in yeast extract to study interactions with cytosolic chaperones
Abstract
1 Introduction
2 Yeast cells used for in vitro translation
3 Yeast extract preparation for in vitro translation
4 Preparation of a plasmid encoding the protein of interest for translation in yeast extract
5 In vitro transcription of the gene of interest
6 In vitro translation reaction in yeast extract
7 Pull down assay
Acknowledgments
References
Section 5 Mitochondrial protein translocation
Chapter Fifteen: In vitro import of mitochondrial precursor proteins into yeast mitochondria
Abstract
1 Introduction
2 In vitro synthesis of mitochondrial precursor proteins
3 In vitro import into isolated yeast mitochondria
Acknowledgments
References
Chapter Sixteen: Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria
Abstract
1 Introduction
2 In vitro synthesis of 35S-radiolabelled precursor proteins
3 Mitochondrial isolation
4 In vitro import assay
5 Monitoring assembly into protein complexes
6 Visualising in vitro import reactions
7 Concluding remarks
Acknowledgements
References
Chapter Seventeen: Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells
Abstract
1 Introduction
2 Mitochondrial protein import: routes and targeting sequences
3 Semi-intact cells
4 Import of proteins into semi-intact cells
5 Notes
6 General considerations
Acknowledgments
References
Chapter Eighteen: The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import
Abstract
1 Introduction
2 Methods
References
Chapter Nineteen: Monitoring mitochondrial protein import by live cell imaging
Abstract
1 Introduction
2 Equipment and materials
3 Protocol
4 Notes
Acknowledgment
References
Chapter Twenty: Quantifying mitochondrial protein import by mePRODmt proteomics
Abstract
1 Introduction
2 Materials
3 Methods
Acknowledgments
References
Chapter Twenty One: Methods to analyze mitochondrial protein translocation in plant mitochondria
Abstract
1 Introduction
2 Plants and tissues
3 Dual-targeting verse mis-targeting
4 The import of precursor proteins into isolated plant mitochondria
5 Protein import assays using freshly isolated mitochondria
6 In vivo protein uptake studies with fluorescent protein tagging
References
Section 6 Mitochondrial gene expression and translation
Chapter Twenty Two: In organello silencing of mitochondrial gene expression
Abstract
1 Introduction
2 Protein carrier preparation
3 HEK293T mitochondrial isolation
4 In organello silencing of mitochondrial gene expression
5 Conclusion
6 Troubleshooting
References
Chapter Twenty Three: Assays to monitor mitochondrial translation
Abstract
1 Introduction
2 Before you begin
3 Materials and equipment
4 Step-by-step method details
5 Expected outcomes
6 Quantification and statistical analysis
7 Advantages
8 Limitations
9 Optimization and troubleshooting
10 Safety considerations and standards
References
Chapter Twenty Four: Analysis of mitochondrial translation using click chemistry
Abstract
Abbreviations
1 Introduction
2 Cell culture
3 L-Homopropargylglycine (HPG) incorporation in living cells
4 HPG click chemistry in isolated mitochondria
5 HPG click chemistry for microscopy imaging
6 High resolution microscopy
7 Trouble shooting
8 Conclusions and outlook
Acknowledgments
References

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Mitochondrial Translocases Part A

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