Mitochondrial Translocases Part B

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