Cover	1
	CONTENTS	6
	Contributors	14
	Preface	18
	Chapter 1. Isolation and Subfractionation of Mitochondria from Animal Cells and Tissue Culture Lines	20
	I. Introduction	21
	II. General Properties of Mitochondrial Preparations	21
	III. Mitochondria from Beef Heart	24
	IV. Mitochondria from Rat Liver	32
	V. Mitochondria from Muscle	38
	VI. Synaptic and Nonsynaptic Mitochondria from Different Rat Brain Regions	39
	VII. Mitochondria from Hamster Brown Adipose Tissue	42
	VIII. Mitochondria from Insect Flight Muscle	43
	IX. Mitochondria from Porcine Adrenal Cortex	43
	X. Mitochondria from Human Platelets	44
	XI. Mitochondria from Fish Liver	47
	XII. Mitochondria from Sea Urchin Egg	47
	XIII. Mitochondria and Kinetoplasts from Protozoa	47
	XIV. Mitochondria and Mitoplasts from Cultured Cells	49
	References	50
	Chapter 2. Isolation and Subfractionation of Mitochondria from the Yeast Saccharomyces cerevisiae	56
	I. Introduction	57
	II. Isolation of Yeast Mitochondria of Different Purity	58
	III. Analysis of Mitochondria Preparations	63
	IV. Purification of Outer Membrane Vesicles from Yeast Mitochondria	67
	References	68
	Chapter 3. Isolation and Subfractionation of Mitochondria from Plants	72
	I. Introduction	73
	II. Growth and Preparation of Plant Material	73
	III. Isolation of Mitochondria by Differential Centrifugation	74
	IV. Density Gradient Purification of Mitochondria	76
	V. Mitochondrial Yield, Purity, Integrity, Storage, and Function	80
	VI. Subfractionation of Mitochondrial Compartments	83
	VII. Proteome Analysis	87
	VIII. Analysis of in Organello Translation Products	89
	IX. Conclusion	91
	References	91
	Chapter 4. Assessing Functional Integrity of Mitochondria in Vitro and in Vivo	94
	I. Introduction	94
	II. In Vitro Assays with Mitochondrial Preparations	95
	III. Mitochondrial Functions within Living Cells	106
	References	112
	Chapter 5. Assaying Mitochondrial Respiratory Complex Activity in Mitochondria Isolated from Human Cells and Tissues	116
	I. Introduction	116
	II. Preparation of Mitochondrial Fractions	117
	III. Spectrophotometric Measurement of the Activities of Individual Complexes I–IV	120
	IV. Linked Assays	131
	V. Applications of Techniques to Diagnostic Investigations	134
	References	135
	Chapter 6. In Vivo Measurements of Respiration Control by Cytochrome c Oxidase and in Situ Analysis of Oxidative Phosphorylation	138
	I. Introduction	139
	II. Measurements of Endogenous Respiration in Intact Cells	139
	III. KCN Titration of COX Activity in Intact Cells	141
	IV. KCN Titration of Integrated COX Activity	145
	V. KCN Titration of Isolated COX Activity	144
	VI. In Situ Analysis of Mitochondrial OXPHOS	145
	References	149
	Chapter 7. Assay of Mitochondrial ATP Synthesis in Animal Cells	152
	I. Introduction	152
	II. Methodological Considerations	154
	III. Experimental Procedures	155
	IV. ATP Synthesis in Cellular Models of Mitochondrial Diseases	160
	References	162
	Chapter 8. Measurement of Membrane Permeability and Permeability Transition of Mitochondria	166
	I. Introduction	166
	II. Procedures	167
	III. Anticipated Results and Pitfalls	175
	References	176
	Chapter 9. Assaying Actin-Binding Activity of Mitochondria in Yeast	178
	I. Introduction	178
	II. Purification of Yeast Actin	179
	III. Sedimentation Assay for Binding of Actin to Mitochondria	184
	IV. Analysis of Mitochondria-Actin Binding	189
	References	191
	Chapter 10. Analysis and Prediction of Mitochondrial Targeting Peptides	194
	I. Introduction	194
	II. What Mitochondrial Targeting Peptides (mTPs) Look Like	195
	III. Automatic Classification of Known mTPs	196
	IV Automatic Identification of Unknown mTPs	197
	V Conclusions	202
	References	203
	Chapter 11. Assaying Protein Import into Mitochondria	208
	I. Introduction	209
	II. Synthesis of Preproteins	209
	III. Standard Protocol for Protein Import into Isolated Mitochondria	215
	IV. Assaying Mitochondrial Localization of Imported Preproteins	218
	V. Energetic Requirements for Preprotein Import	223
	VI. Assaying Import Pathways	224
	VII. Assaying Protein Assembly by Blue-Native Electrophoresis	227
	VIII. Import of Purified Recombinant Proteins into Mitochondria	228
	References	232
	Chapter 12. Analysis of Protein–Protein Interactions in Mitochondria by Coimmunoprecipitation and Chemical Cross-Linking	236
	I. Introduction	236
	II. General Considerations	237
	III. Immunoprecipitation	238
	IV. Coimmunoprecipitation	241
	V. Cross-Linking	244
	VI. Summary	247
	References	248
	Chapter 13. Blue-Native Gels to Isolate Protein Complexes from Mitochondria	250
	I. Introduction	250
	II. Materials and Methods	251
	III. Applications	257
	IV. Outlook	261
	References	261
	Chapter 14. Application of Electron Tomography to Mitochondrial Research	264
	I. Introduction	264
	II. Preliminary Considerations for Application of Electron Tomography	267
	III. Methodology	268
	References	274
	Chapter 15. Epitope Tagging and Visualization of Nuclear-Encoded Mitochondrial Proteins in Yeast	276
	I. Introduction	276
	II. Single-step Modification of Nuclear-Encoded Mitochondrial Genes in Yeast: An Overview	277
	III. Generation and Analysis of the Modified Gene Product: Technical Considerations	277
	IV. Visualizing Yeast Mitochondria by Immunostaining	284
	References	293
	Chapter 16. Targeting of Green Fluorescent Protein to Mitochondria	296
	I. Introduction	296
	II. Mitochondrial Green Fluorescent Protein (GFP) Fusion Markers	297
	III. General Concerns about Targeting of GFP Markers into Mitochondria	300
	IV. Conclusions	301
	References	302
	Chapter 17. Assessment of Mitochondrial Membrane Potential in Situ Using Single Potentiometric Dyes and a Novel Fluorescence Resonance Energy Transfer Technique	304
	I. Introduction	304
	II. Single Potentiometric Dyes	307
	III. Novel Fluorescence Resonance Energy Transfer Assay for ??m	313
	References	325
	Chapter 18. Optical Imaging Techniques (Histochemical, Immunohistochemical, and in Situ Hybridization Staining Methods) to Visualize Mitochondria	330
	I. Introduction	331
	II. Histochemistry	332
	III. Immunohistochemistry	336
	IV. In Situ Hybridization and Single-Fiber Polymerase Chain Reaction (PCR)	340
	V. Histochemical Methods	345
	VI. Immunohistochemical Methods	345
	VII. Molecular Genetic Methods	347
	References	348
	Chapter 19. Visualization of Mitochondrial Movement in Yeast	352
	I. Introduction	352
	II. Yeast Cell Growth and Synchronization	354
	III. Vital Staining of Yeast Mitochondria	356
	IV. Preparation of Growth Chamber	362
	V. Image Acquisition	363
	VI. Analysis and Quantitation of Movement	365
	References	368
	Chapter 20. Targeting of Reporter Molecules to Mitochondria to Measure Calcium, ATP, and pH	372
	I. Introduction	373
	II. Calcium	376
	III. ATP	384
	IV. pH	391
	V. Conclusion	395
	References	396
	Chapter 21. Genetic Transformation of Saccharomyces cerevisiae Mitochondria	400
	I. Introduction	401
	II. Important Features of Saccharomyces cerevisiae Mitochondrial (MT) Genetics	401
	III. Delivery of DNA to the Mitochondrial Compartment of rho0 Cells and Detection of Mitochondrial Transformants	403
	IV. Strategies for Gene replacement in S. cerevisiae mtDNA	407
	V. Transformation of rho+ Cells with Plasmids or Linear DNA Fragments	411
	VI. Concluding Remarks	413
	References	413
	Chapter 22. Transmitochondrial Technology in Animal Cells	416
	I. Introduction	416
	II. Generation of p0 Cells	418
	III. Generation of Transmitochondrial Cybrids Using p0 Cells as Nuclear Donors	420
	IV. Generation of Transmitochondrial Cybrids Using p+ Cells Treated with Rhodamine 6G as Nuclear Donors	425
	V. Generation of Transmitochondrial Hybrid Cells by Microcell-Mediated Chromosome and mtDNA Transfer	426
	VI. Manipulating Heteroplasmy	428
	References	429
	Chapter 23. Diagnostic Assays for Defects in Mitochondrial DNA Replication and Transcription in Yeast and Human Cells	432
	I. Introduction	432
	II. Diagnosis of Mitochondrial (mt)DNA Replication Defects in Yeast and Human Cells	433
	III. Analyzing Mitochondrial Transcripts in Vivo	438
	IV. Additional Considerations	442
	References	444
	Chapter 24. Analysis of Mitochondrial Translation Products in Vivo and in Organello in Yeast	448
	I. Introduction	448
	II. Labeling of Mitochondrial Translation Products in Vivo	450
	III. Labeling of Mitochondrial Translation Products in Organello	450
	IV. Use of Mitochondrial Translation for the Study of Various Aspects of Mitochondrial Biogenesis	451
	V. Analysis of Mitochondrial Translation Products by Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis	454
	VI. Discussion	455
	References	456
	Chapter 25. Numerical Methods for Handling Uncertainty in Microarray Data: An Example Analyzing Perturbed Mitochondrial Function in Yeast	458
	I. Introduction	458
	II. Quantitative Methods in Microarray Research	459
	III. Application of Microarrays to the Study of Cellular Responses to Perturbed Mitochondrial Function	465
	References	470
	APPENDICES	472
	APPENDIX 1. Basic Properties of Mitochondria	472
	APPENDIX 2. Linearize Maps of Circular Mitochndrial Genomes from	476
	APPENDIX 3. Mitochondrial Genetic Codes in Various Organisms	480
	APPENDIX 4. Gene Products Present in Mitochndria of Yeast and Animal Cells	482
	APPENDIX 5. Direct and Indirect Inhibitors of Mitochndrial ATP Synthesis	502
	Index	516
	Volumes in Series	532