Mitochondria

Cover

Contentsv

Contributorsxvii

Prefacexxiii

Part I: Isolation and Subfractionation of Mitochondria1

Chapter 1: Isolation and Subfractionation of Mitochondria from Animal Cells and Tissue Culture Lines3

I. Introduction4

II. General Features of Mitochondrial Preparations5

A. Cell Rupturing by Mechanical and/or Chemical Means and Resuspension in Isolation Medium5

B. Differential Centrifugation7

C. Storage8

D. Criteria of Purity and Intactness8

III. Mitochondria from Beef Heart8

A. Small-Scale Preparation9

B. Preparation of Coupled Submitochondrial Particles11

C. Broken Submitochondrial Particles12

D. Keilin-Hartree Heart Muscle Preparation13

E. Cytochrome c-Depleted and Cytochrome c-Reconstituted Mitochondria and ETPH14

F. CoQ-Depleted and CoQ-Reconstituted Mitochondria15

IV. Mitochondria from Rat Liver17

A. Standard Preparation18

B. Gradient-Purified Rat Liver Mitochondria18

C. Liver SMP20

D. Mitochondria from Rat Hepatocytes20

E. Isolation of Mitochondria from Frozen Tissues20

F. Preparation of Rat Liver Mitoplasts21

G. Phospholipid-Enriched Mitoplasts and SMP with or Without Excess CoQ22

V. Mitochondria from Skeletal Muscle23

VI. Synaptic and Nonsynaptic Mitochondria from Different Rat Brain Regions26

VII. Mitochondria from Kidney and Testis29

VIII. Mitochondria from Hamster Brown Adipose Tissue29

IX. Mitochondria from Insect Flight Muscle30

X. Mitochondria from Porcine Adrenal Cortex30

XI. Mitochondria from Human Platelets31

A. Crude Mitochondrial Membranes31

B. Coupled Mitochondrial Particles from Platelet Mitochondria32

XII. Mitochondria from Fish Liver32

XIII. Mitochondria from Sea Urchin Eggs33

XIV. Mitochondria and Kinetoplasts from Protozoa33

XV. Mitochondria from Fish Erythrocytes35

XVI. Mitochondria from Caenorhabditis elegans35

XVII. Mitochondria from Drosophila35

XVIII. Mitochondria and Mitoplasts from Cultured Cells36

Acknowledgments39

References39

Chapter 2: Purification and Subfractionation of Mitochondria from the Yeast Saccharomyces cerevisiae45

I. Introduction45

II. Isolation of Mitochondria46

A. Growth of Yeast Cells49

B. Isolation of Crude Mitochondria49

C. Purification of Crude Mitochondria Using Continuous Nycodenz Gradient Centrifugation51

III. Analysis of Isolated Mitochondria53

A. Assessing the Purity of Mitochondrial Preparations53

B. Determining the Integrity of Mitochondrial Preparations55

C. Assessment of Protein Localization to Mitochondria and to Subcompartments in the Organelle58

D. Assessment of the Disposition of Proteins on Mitochondrial Membranes59

IV. Isolation of Mitochondrial Outer Membranes, Contact Sites, and Inner Membranes61

Acknowledgments63

References63

Chapter 3: Isolation and Subfractionation of Mitochondria from Plants65

I. Introduction66

II. Growth and Preparation of Plant Material66

III. Isolation of Mitochondria67

IV. Density Gradient Purification of Mitochondria69

A. Sucrose69

B. Percoll69

C. Mini-Mitochondrial. Preparations for Screening Transgenic Lines71

D. Cell Culture/Callus Mitochondria Preparation72

V. Mitochondrial Yield, Purity, Integrity, Storage, and Function73

A. Yield Calculations73

B. Purity Determinations74

C. Integrity Determinations74

D. Storage75

E. Assays of Mitochondria Function75

VI. Subfractionation of Mitochondrial Compartments77

A. Separation of Inner Mitochondrial Membrane, Outer Mitochondrial Membrane, Matrix, and Intermembra77

B. Separation of Mitochondrial Inner Membrane Components by MonoQ78

C. Isoelectric Focusing and SDS-PAGE80

D. BN-PAGE of Intact ETC Components81

VII. In Organello Translation Analysis83

VIII. Proteome Analysis85

A. Experimental Analysis of Mitochondrial Proteomes85

B. Prediction of Mitochondrial Proteomes86

IX. Conclusion87

References87

Part II: Biochemical Assays of Mitochondrial Activity91

Chapter 4: Biochemical Assays of Respiratory Chain Complex Activity93

I. Introduction93

II. Materials and Methods96

A. Sample Preparation96

B. Enzyme Assays101

III. Interpreting Results of RC Enzymology114

Acknowledgments116

References116

Chapter 5: Polarographic Assays of Respiratory Chain Complex Activity121

I. Introduction121

II. Measurements of Endogenous Respiration in Intact Cells122

A. Media and Reagents122

B. Procedure122

C. Comments123

III. KCN Titration of COX Activity in Intact Cells124

A. Principle124

B. Media and Reagents125

C. Procedure125

D. Analysis of Data127

IV. In Situ Analysis of Mitochondrial OXPHOS128

A. Media and Reagents129

B. Digitonin Titration129

C. P/O Ratio Assay129

D. Analysis of Data130

Acknowledgments132

References132

Chapter 6: Optical Imaging Techniques (Histochemical, Immunohistochemical, and In Situ Hybridization135

I. Introduction135

II. Histochemistry137

A. Succinate Dehydrogenase138

B. Cytochrome c Oxidase140

III. Immunohistochemistry142

A. Immunolocalization of nDNA- and mtDNA-Encoded Subunits of the Respiratory Chain in Frozen Samples142

B. Immunolocalization of mtDNA on Frozen Samples145

C. Immunolocalization of Mitochondrial Proteins on Paraffin-Embedded Samples146

IV. In Situ Hybridization to mtDNA148

Acknowledgments151

References151

Chapter 7: Assay of Mitochondrial ATP Synthesis in Animal Cells and Tissues155

I. Introduction155

II. ATP Synthesis Assays156

III. Methodological Considerations157

A. Cell Permeabilization (Detergent Titration in Cultured Cells)157

B. ATP Detection by Luciferase-Luciferin157

C. Specificity of the Assay158

IV. Experimental Procedures160

A. Measurement of ATP Synthesis in Cultured Cells160

B. ATP Synthesis in Mitochondria Isolated from Animal Tissues162

V. Measurement of High-Energy Phosphates in Animal Tissue and Cultured Cells by HPLC165

A. Apparatus and Reagents166

B. Preparation of Biological Samples166

C. Chromatography167

D. Standard Curves167

E. Measurement of Creatine, Phosphocreatine, and Phosphorylated Nucleotides168

Acknowledgments169

References169

Chapter 8: Measurement of the Ratio of Lactate to Pyruvate in Skin Fibroblast Cultures173

I. Introduction173

II. Principle174

III. Procedure175

A. Sample Preparation175

B. Determination of Lactate176

C. Determination of Pyruvate176

IV. Results177

References178

Chapter 9: Assays of Fatty Acid beta-Oxidation Activity179

I. Introduction179

II. Metabolite Measurements183

A. Acylcarnitine Profiles and Total and Free Carnitine Levels183

B. Measurement of Urine Organic Acids and Acylglycine184

III. Enzyme and Transporter Assays186

A. Carnitine Uptake186

B. Carnitine Cycle186

C. Intramitochondrial Enzymes189

IV. Metabolic Flux Studies193

A. Whole-Flux Measurement193

B. Metabolite Accumulation Assays195

V. Discussion195

References195

Chapter 10: Biochemical Assays for Mitochondrial Activity: Assays of TCA Cycle Enzymes and PDHc199

I. Introduction199

II. Pyruvate Dehydrogenase Complex202

A. Pyruvate Dehydrogenase Assays202

B. LAD Assay203

III. Citrate Synthase207

IV. Aconitase209

V. Isocitrate Dehydrogenase209

VI. alpha-Ketoglutarate Dehydrogenase Complex210

VII. Succinyl CoA Synthetase212

VIII. Succinate Dehydrogenase213

IX. Fumarase214

X. Malate Dehydrogenase217

XI. Conclusion217

Acknowledgments217

References217

Chapter 11: Assays of Cardiolipin Levels223

I. Introduction223

II. Molecular Species of Cardiolipin225

III. Cardiolipin Analysis by TLC226

A. Overview226

B. Protocol for TLC with Phosphorus Assay228

C. Protocol for TLC with Fatty Acid Assay230

IV. Cardiolipin Analysis by HPLC230

A. Overview230

B. Protocol for Nonderivatized Cardiolipin232

C. Protocol for Fluorescence-Labeled Cardiolipin232

V. Cardiolipin Analysis by MS234

A. Overview234

B. Method235

VI. Applications of Cardiolipin Measurements236

Acknowledgments238

References238

Chapter 12: Measurement of VDAC Permeability in Intact Mitochondria and in Reconstituted Systems241

I. Introduction241

II. Determining the Permeability of the Mitochondrial Outer Membrane to Metabolites243

A. Rationale243

B. Assay of Outer Membrane Intactness243

C. Methods243

D. Controls252

III. VDAC Activity After Reconstitution into Phospholipid Membranes253

A. Rationale253

B. Purification of VDAC253

C. Reconstitution of VDAC into Planar Membranes254

D. Electrophysiological Recordings256

IV. Summary259

Acknowledgments259

References259

Chapter 13: Methods for Studying Iron Metabolism in Yeast Mitochondria261

I. Introduction261

II. Determination of Cellular Fe/S Protein Formation and Heme Synthesis263

A. Determination of Cellular Fe/S Cluster Formation by Radiolabeling of Yeast Cells In Vivo264

B. Determination of Cellular Heme Levels by Radiolabeling of Yeast Cells In Vivo267

C. Determination of Fe/S Cluster Formation in Isolated Mitochondria In Vitro268

D. Analysis of Fe/S Cluster Formation In Vitro Using Recombinant Ferredoxins270

III. Determination of Mitochondrial Iron Contents272

A. Bathophenantroline Assay273

B. Nitro-PAPS Assay273

IV. Reporter Assays for Analysis of Iron-Dependent Gene Expression in S. cerevisiae274

A. Promoter Assay Based on Green Fluorescent Protein276

B. Promoter Assays Based on Luciferase Reporters276

Acknowledgments279

References279

Part III: Assays for Mitochondrial Respiratory Activity and Permeability in Living Cells281

Chapter 14: Imaging of Mitochondrial Polarization and Depolarization with Cationic Fluorophores283

I. Introduction283

II. Quantitative Imaging of DeltaPsi with Fluorescent Cations284

A. Nernstian Distribution of Cationic Fluorophores284

B. Cellular Loading of Potential-Indicating Fluorophores285

C. Image Acquisition and Processing285

D. Nonideal Characteristics of Fluorophores289

III. Visualization of Depolarized Mitochondria289

A. Covalent Adduct Formation by MitoTracker Probes289

B. FRET Between Cationic Fluorophores290

C. FRET Between MitoTracker Green FM and TMRM291

D. Use of FRET to Distinguish Depolarized from Polarized Mitochondria292

IV. Conclusion293

Acknowledgments294

References294

Chapter 15: Biosensors for the Detection of Calcium and pH297

I. Introduction297

II. Targeting Strategies and Transfection298

A. Targeting to the Mitochondrial Matrix298

B. Targeting to the Mitochondrial Intermembrane Space299

III. Calcium Reporters300

A. Mitochondrial Calcium Measurements Using Aequorin301

B. Procedure305

C. Mitochondrial Calcium Measurements Using GFP311

D. Procedure316

IV. pH Reporters316

A. Mitochondrial pH-Sensitive Fluorescent Proteins317

B. Procedure321

V. Conclusions321

Acknowledgments321

References322

Chapter 16: Measurement of Membrane Permeability and the Permeability Transition of Mitochondria327

I. Introduction327

II. Procedures328

A. Mitochondrial Alterations in Intact Cells328

B. Cytometric Analysis: Lipophilic Cationic Dyes328

References338

Chapter 17: Luciferase Expression for ATP Imaging: Application to Cardiac Myocytes341

I. Introduction341

II. ATP Homeostasis in the Heart343

III. Measurement of Mitochondrial and Cytosolic ATP in Cardiac Myocytes343

IV. Measurement of ATP in Subcellular Compartments by Luminometery346

A. Luciferase Vectors347

B. Cell Transfection and ATP Assays348

C. ATP Standards350

Acknowledgments351

References351

Part IV: Oxidative Stress Measurements353

Chapter 18: Measurement of Reactive Oxygen Species in Cells and Mitochondria355

I. Introduction355

II. Measurement of ROS in Cells357

A. CL-Based Characterization of ROS Generated by HX/XO and Phorbol Myristate Acetate (PMA)-Stimulate357

B. Electron Spin Resonance Spectroscopy361

C. Fluorescence Dye Techniques for the Determination of Cellular ROS363

D. The Determination of ROS Using HE and FACS Analyses366

E. Spectrophotometric Methods for Determination of ROS367

III. Measurement of ROS in Mitochondria369

A. Isolation of Mitochondria from Cells and Tissue369

B. Measurement of Mitochondrial ROS (H2O2)370

C. Measurement of Mitochondrial ROS (O2bull-)371

IV. General Problems Associated with the Measurement of ROS372

V. Conclusions373

Acknowledgments374

References374

Chapter 19: Measurements of the Antioxidant Enzyme Activities of Superoxide Dismutase, Catalase, and379

I. Introduction379

II. Experimental Procedures380

A. Sample Preparation380

B. SOD Activity Assay381

C. GPx Activity Assay386

D. Catalase Activity Assay389

Acknowledgments391

References391

Chapter 20: Methods for Measuring the Regulation of Respiration by Nitric Oxide395

I. Introduction396

A. Basic Concepts of Metabolic Control396

II. Apparatus398

A. Classical Closed System Respirometer398

B. Open-Flow Respirometer400

III. Calibration of the NObull Electrode401

A. Preparing NObull Stock Solutions for Electrode Calibration401

B. NObull Electrode Calibration401

C. Effects of O2 on NObull Electrode Calibration402

D. NObull Release from NObull Donor Compounds404

IV. NObull Threshold Measurement406

A. Threshold Measurements in Isolated Mitochondria406

B. Altered Thresholds in Disease408

C. Measurement of NObull Thresholds in Cells408

V. Open-Flow Respirometry411

VI. An In Vitro Model of Ischemia/Reperfusion Injury413

VII. Conclusion414

Acknowledgments415

References415

Chapter 21: Methods for Determining the Modification of Protein Thiols by Reactive Lipids417

I. Introduction417

A. Formation of Reactive Lipids in Physiology and Pathology417

B. Electrophilic Lipids as Reactive Lipid Mediators418

C. Reactivity of Electrophilic Lipids with Protein Thiols and the Role of Mitochondria419

II. Rationale419

III. Methods421

A. Biotin Labeling of Free Protein Thiols Using Biotinylated Iodoacetamide421

B. Synthesis of a Biotinylated Electrophilic Lipid422

C. Preparation and Calibration of Biotinylated Cytochrome c and Other Proteins425

D. Blotting and Image Analysis429

IV. Discussion432

Acknowledgments433

References433

Part V: Mitochondrial Genes and Genes and Gene Expression435

Chapter 22: Detection of Mutations in mtDNA437

I. Introduction438

II. Large-Scale Rearrangements of mtDNA439

III. Detecting mtDNA Rearrangements440

IV. Details of Southern Blot Hybridization Analysis443

A. DNA Preparation443

B. Probe Preparation443

C. Labeling of the Probe444

D. Preparation of Restriction Enzyme Digest444

E. Agarose Gel Elect445

F. DNA Transfer445

G. Hybridization445

H. Washing the Membrane446

I. Film Developing446

V. Depletion of mtDNA447

A. Southern Blot Hybridization Analysis to Detect mtDNA Depletion447

B. Quantitation of mtDNA by Real-Time PCR448

VI. Point Mutations in mtDNA449

A. Detecting mtDNA Point Mutations by PCR/RFLP Analysis451

B. Quantification of Heteroplasmy Level by the Amplification-Refractory Mutation System and Quantita456

C. Detection of Unknown Mutations in mtDNA459

Acknowledgments460

References460

Chapter 23: Diagnostic Assays for Defects in mtDNA Replication and Transcription in Yeast and Humans465

I. Introduction465

II. Diagnosis of mtDNA Replication Defects in Yeast and Human Cells466

A. Fundamental Aspects of mtDNA Replication466

B. Methods for Detecting mtDNA and Measuring Copy Number467

III. Analyzing Mitochondrial Transcripts In Vivo472

A. Mitochondrial Transcription Initiation472

B. Standard Assays for Mitochondrial Transcription473

IV. Additional Considerations477

Acknowledgment477

References477

Chapter 24: Microdissection and Analytical PCR for the Investigation of mtDNA Lesions481

I. Introduction481

II. Microdissection and DNA Purification482

A. Tissue Preparation and Sectioning482

B. Microdissection Techniques483

C. Laser Microdissection484

D. DNA Purification485

E. PCR487

III. Analytical PCR for Investigation of mtDNA in Microdissected Samples487

A. Common Strategies for Improving PCR Signal Strength from Microdissected Samples487

B. RELP of know Polymorphisms and Mutations490

C. Sample Protocol490

D. mtDNA Sequencing493

E. Quantification of mtDNA Content493

F. Detection of DeltamtDNA Species495

G. Quantification of DeltamtDNA Abundance496

IV. Concluding Remarks498

Acknowledgments498

References499

Chapter 25: Transmitochondrial Technology in Animal Cells503

I. Introduction504

II. Generation of Nuclear Donors504

A. Generation of rho0 Cells504

B. rho+ Cells Treated with Rhodamine 6G as Nuclear Donors507

III. Generation of mtDNA Donors510

A. Cultured Cells as mtDNA Donors Enucleated with Cytochalasin B and Centrifugation (Cytoplasts)510

B. Platelets as mtDNA Donors512

C. Synaptosomes as mtDNA Donors512

D. Chemical Enucleation to Produce mtDNA Donors513

IV. Generation of Transmitochondrial Cybrids514

A. Using Enucleated Cells as mtDNA Donors and rho0 Cells as Nuclear Donors514

B. Using.Platelets as Mitochondrial Donors515

C. Using rho+ Cells Treated with R6G as Nuclear Donors516

D. Using Synaptosomes as Mitochondrial Donors516

E. Using Cells Chemically Enucleated with Actinomycin D as Mitochondrial Donors516

F. Generation of Transmitochondrial Hybrid Cells by Microcell-Mediated Chromosome and mtDNA Transfer517

V. Manipulating Heteroplasmy519

A. Manipulating Heteroplasmy by the Use of EtBr519

B. Manipulating Heteroplasmy by Delivering Restriction Endonucleases to Mitochondria519

Acknowledgments520

References520

Chapter 26: Genetic Transformation of Saccharomyces.cerevisiae and Chlamydomonas reinhardtii Mitocho525

I. Introduction526

II. Important Features of S. cerevisiae and C. reinhardtii Mitochondrial Genetics527

A. Phenotypes Associated with Mitochondrial Gene Expression527

B. Novel Mitochondrial Genes Conferring Useful Mitochondrial Phenotypes in S. cerevisiae528

C. Replication of mtDNA and Mitochondrial Deletion Mutants529

D. Recombination and Segregation of mtDNA530

III. Delivery of DNA to the Mitochondrial Compartment of rho0 Cells and Detection of Mitochondrial T532

A. Overview of Transformation Procedure532

B. Experimental Details for Transformation and Identification of Mitochondrial Transformants533

IV. Strategies for Gene Replacement in S. cerevisiae mtDNA538

A. Integration of Altered mtDNA Sequences by Homologous Double Crossovers538

B. Experimental Details for Mating and Isolation of Recombinant Cytoductants539

C. Streamlining the Integration of Multiple Mutations in a Short Region by Use of rho+ Recipients Co540

D. Experimental Details for Identification of Nonrespiring Cytoductants by Marker Rescue540

V. Transformation of rho+ Cells with Plasmids or Linear DNA Fragments542

VI. Concluding Remarks544

Acknowledgments544

References544

Chapter 27: Generation of Transmitochondrial Mice: Development of Xenomitochondrial Mice to Model Ne549

I. Introduction549

II. Spontaneous and Induced Models of Mitochondrial Disease550

III. Transgenic Models552

IV. Introduction of Mutant mtDNA into Mitochondria552

A. Use of Transfected rho0 Cells as Intermediate Mitochondrial Carriers in the Production of Mouse M553

B. Embryonic Stem Cell Technology554

V. The First Transmitochondrial Mice555

A. mtDNA Injection Versus ES Cell-Derived Models558

B. Xenomitochondrial Mice560

C. Neurodegenerative Disorders563

VI. Summary/Future Directions564

Acknowledgments564

References565

Chapter 28: In Vivo and In Organello Analyses of Mitochondrial Translation571

I. Introduction571

II. Rationale573

III. Methods576

A. In Vivo Mitochondrial Protein Synthesis576

B. In Organello Protein Synthesis580

C. Complementary Analyses: In Organello Aminoacylation Assay581

IV. Materials583

V. Discussion584

VI. Summary585

Acknowledgments585

References585

Part VI: Assays for Mitochondrial Morphology and Motility589

Chapter 29: Visualization of Mitochondria in Budding Yeast591

I. Introduction592

II. Use of Vital Dyes to Stain Yeast Mitochondria592

A. DNA-Binding Dyes592

B. Lipophilic Membrane Potential-Sensing Dyes594

C. Staining Protocol595

III. Use of Immunostaining to Detect Mitochondria in Fixed Yeast Cells596

A. Pretreatment of Antibodies with Yeast Cell Walls596

B. Yeast Cell Growth, Fixation, and Cell Wall Removal598

C. Preparation of a Staining Chamber601

D. Immobilization of Fixed Spheroplasts on Coverslips and Incubation with Primary and Secondary Anti602

IV. Ectopic Expression of Mitochondria-Targeted Fluorescent Fusion Proteins604

A. Transformation of Yeast with Plasmid-Borne Targeted FPs605

B. Validation of Targeted FPs607

V. Tagging Endogenous Proteins with Fluorescent Proteins607

A. Vectors for PCR-Mediated Tagging of Chromosomal Genes608

B. PCR Amplification of Insertion Cassette608

C. Transformation of Yeast with the Amplified Insertion Cassette611

D. Validation of Tagging and Gene Function612

E. Marker Excision by Cre Recombinase612

VI. Strategies for Visualization of Yeast Mitochondria614

A. Defining Imaging Needs614

B. Imaging Technologies616

C. Equipment for Imaging619

VII. Methods for Imaging of Mitochondria in Living Yeast Cells620

Acknowledgments624

References624

Chapter 30: Visualization and Quantification of Mitochondrial Dynamics in Living Animal Cells627

I. Introduction628

II. Labeling of Mitochondria in Living Animal Cells631

A. Fluorescent Labeling of Mitochondria Using Mitochondrial Dyes and Fluorescent Proteins631

III. Probing Interactions Between Mitochondria and the Cytoskeleton and Molecular Motors in Living A634

A. Visualization of the Cytoskeleton in Living Animal Cells634

B. Use of Cytoskeletal Inhibitors in Living Animal Cells635

C. Inhibition of Microtubule-Based Molecular Motors in Living Animal Cells635

IV. Image Acquisition639

A. Equipment639

V. Preparation of Cells645

A. Isolation, Culture, and Transfection of Primary Cortical Neurons645

B. Culture and Transfection of N2A Neuroblastoma Cells and CV-1 Cells647

VI. Time-Lapse Recordings648

VII. Analysis of Mitochondrial Motility650

A. Software650

B. Analysis of Total Mitochondrial Movement650

C. Kymograph Analysis651

D. Full Quantitative Analysis of Mitochondrial Motility653

VIII. Analysis of Mitochondrial Morphology660

A. Analysis of Morphology of Individual Mitochondria and Mitochondrial Fusion and Fission660

B. Analysis of Mitochondrial Branching and Networks664

IX. Appendix A666

A. Total_Motility666

X. Appendix B670

A. SlopeToVelocity_670

Acknowledgments673

References673

Chapter 31: Cell-Free Assays for Mitochondria-Cytoskeleton Interactions683

I. Introduction683

II. In Vitro Reconstitution of Microtubule-Based Mitochondrial Transport684

A. Preparation of Motility Assay Components685

B. Motility Assays694

C. Analysis of Mitochondrial Motility696

III. Sedimentation Assay for Binding of Actin to Mitochondria696

A. Reagents698

B. Binding Assay700

C. Salt Treatment of Mitochondria702

D. Analysis of Mitochondria-Actin Binding703

Acknowledgments705

References705

Chapter 32: In Vitro Assays for Mitochondrial Fusion and Division707

I. Introduction707

II. Mitochondrial Fusion In Vitro708

A. Plasmids709

B. Cell Culturing and Preparation of Fusion Competent Mitochondria710

C. Fusion Reactions711

D. Analysis of Fusion In Vitro712

III. Analysis of Dynamin-Related GTPase Activity In Vitro712

A. Continuous GTPase Assay714

B. Protein Purification and Storage715

C. Reagents715

D. Procedure716

E. Data Analysis717

Acknowledgments718

References718

Part VII: Methods to Determine Protein Localization to Mitochondria721

Chapter 33: Electrophoretic Methods to Isolate Protein Complexes from Mitochondria723

I. Introduction723

II. Materials and Methods725

A. Chemicals725

B. First Dimension: Blue Native Electrophoresis725

C. First Dimension: Clear Native Electrophoresis730

D. Second Dimension: Modified Blue Native Electrophoresis731

E. Second or Third Dimension: SDS-PAGE732

III. Applications733

A. Protein Complexes from Mitochondria and Chloroplasts734

B. Isolation of Protein Complexes from Tissue Homogenates and Cell Lines738

C. Purification of Partially Purified Protein Complexes Using BN-PAGE738

IV. Outlook738

Acknowledgment739

References739

Chapter 34: Analysis of Protein-Protein Interactions in Mitochondria743

I. Introduction743

II. Rationale744

III. Methods745

A. Coimmunoprecipitation746

B. Copurification Using Affinity Tags751

C. Cross-Linking753

Acknowledgments757

References757

Chapter 35: Analysis and Prediction of Mitochondrial Targeting Signals761

I. Introduction761

II. Overview on Protein Translocation into Mitochondria762

III. Mitochondrial-Targeting Signals764

A. Matrix-Targeting Signal (Presequence)764

B. Internal Targeting Signals768

IV. Bioinformatics Tools to Predict Mitochondrial Targeting Signals772

A. TargetP772

B. PSORT II773

C. MITOPRED773

D. MitoProt II773

E. Predotar773

V. Experimental Analysis of Mitochondrial-Targeting Signals774

A. Construction of Hybrid Proteins774

B. Mutagenesis Approach774

C. Peptide Library Scan775

D. Replacing of the Putative Signal with Known Mitochondrial-Targeting Sequences775

VI. Summary776

Acknowledgments776

References776

Chapter 36: Import of Proteins into Mitochondria783

I. Introduction783

II. In Vitro Synthesis of Precursor Proteins785

A. RNA Preparation and In Vitro Transcription785

B. In Vitro Translation788

III. The Import Reaction789

A. Mitochondrial Isolation789

B. Standard Import Reaction into Isolated Yeast Mitochondria790

C. Time Course Experiment791

IV. Steps to Resolve the Location of Imported Mitochondrial Precursors794

A. Protease Protection Assays794

B. Mitochondrial Swelling795

C. Mitochondrial Solubilization for Protease Treatment796

D. Is the Newly Imported Mitochondrial Protein Soluble, Peripherally Membrane Associated, or Integra796

V. Assaying Protein Complex Assembly by BN-PAGE797

VI. Resolving Protein Complex Constituents798

A. Antibody Shift and Antibody Depletion Assays798

B. Cross-Linking After Import799

VII. Assaying Protein Import in Yeast Mutants801

VIII. Concluding Remarks802

Acknowledgments803

References803

Part VIII: Appendices807

Appendix 1: Basic Properties of Mitochondria809

Appendix 2. Direct and Indirect Inhibitors of Mitochondrial ATP Synthesis813

Appendix 3: Linearized Maps of Circular Mitochondrial Genomes from Representative Organisms827

Appendix 4: Mitochondrial Genetic Codes in Various Organisms831

Appendix 5: Gene Products Present in Mitochondria of Yeast and Animal Cells835

Appendix 6: Changes in the Mitochondrial Transcriptome and Proteome Under Various Stresses and Growt877

Index889

Volumes in Series913