Mitochondrial Function, Part B (eBook)

Front Cover 1
Mitochondrial Function, Part B: Mitochondrial Protein Kinases, Protein Phosphatases and Mitochondrial Diseases 4
Copyright Page 5
Contents 6
Contributors 16
Preface 26
Methods in Enzymology 28
Section 1: Defining the Mitochondrial Proteome, Including Phosphorylated, Acetylated, and Palmitoylated Proteins 56
Chapter 1: The Mitochondrial Proteome Database: MitoP2 58
1. Introduction 59
2. Yeast, Human, and Mouse Proteome Information 61
3. Mitochondrial Reference Set, Candidates, and SVM Prediction Score 63
4. Integrated Genome-Wide Approaches 64
5. Other Search Options: Restrictions to Functional Categories, Gene Locus and Disease-Causing Proteins 67
6. Output Lists and Practical Examples 69
References 72
Chapter 2: Predicting Proteomes of Mitochondria and Related Organelles from Genomic and Expressed Sequence Tag Data 76
1. Introduction 77
2. Data Preparation 83
3. Screening Large Datasets for Mitochondrial Proteins 84
4. Detailed Analysis 88
5. Comparison of Methods 96
Acknowledgments 99
References 99
Chapter 3: Proteome Characterization of Mouse Brain Mitochondria Using Electrospray Ionization Tandem Mass Spectrometry 104
1. Introduction 105
2. Analysis of Mitochondrial Proteome by Two-Dimensional PAGE and LC-MS Techniques 106
3. Selective Proteome Enrichment by Capillary Isotachophoresis (CITP)-Based Separations 106
4. Analysis of Mouse Brain Mitochondria Using the CITP-Based Proteome Platform 107
5. Proteome Characterization of Mouse Brain Mitochondria 110
References 115
Chapter 4: 32P Labeling of Protein Phoshorylation and Metabolite Association in the Mitochondria Matrix 118
1. Introduction 119
2. Methods 120
3. Results and Discussion 127
4. Summary 133
References 134
Chapter 5: Selective Enrichment in Phosphopeptides for the Identification of Phosphorylated Mitochondrial Proteins 136
1. Introduction 137
2. Off-Line Phosphopeptide Enrichment Methods 139
3. On-Line 2D-LC Phosphopeptide Enrichment Methods 145
4. Mass Spectrometry-Based Methods 147
5. Quantitative Phosphoproteomics 149
References 149
Chapter 6: Post-translational Modifications of Mitochondrial Outer Membrane Proteins 152
1. Introduction 153
2. Isolation of Mitochondria and Mitochondrial Membranes 154
3. Detection of Post-translational Modifications Using One-dimensional Gel Electrophoresis 156
4. Focused Proteomics Using Immunological Approaches 160
5. Shotgun Methods for Identification of Post-translational Modifications 164
6. Conclusion 166
Acknowledgments 166
References 167
Chapter 7: Analysis of Tyrosine-Phosphorylated Proteins in Rat Brain Mitochondria 172
1. Introduction 173
2. Identification of Src Tyrosine Kinase Substrates in Rat Brain Mitochondria 174
3. Mass Spectrometric Analysis of Tyrosine-Phosphorylated Peptides in Mitochondria 180
4. Bioinformatic Tools to Analyze the Potential Role of Tyrosine Phosphorylation 186
Acknowledgment 189
References 189
Chapter 8: Acetylation of Mitochondrial Proteins 192
1. Introduction 193
2. Purification of Enzymatically Active SIRT3 193
3. SIRT3 Enzymatic Deacetylation Assay 196
4. Detection of Acetylated Proteins in Mitochondria 199
5. Conclusion 201
References 202
Chapter 9: Non-radioactive Detection of Palmitoylated Mitochondrial Proteins Using an Azido-Palmitate Analogue 204
1. Introduction 205
2. Synthesis of 12-Azidododecanoate and 14-Azidotetradecanoate 208
3. Synthesis of Tagged Phosphine Probes 210
4. Preparation of Azido-Fatty Acids and Tagged Phosphine Stock Solutions 210
5. Production of 14-Azidotetradecanoyl-CoA Derivatives 211
6. In Vitro Labeling of Proteins with 14-Azidotetradecanoyl-CoA and Tagged Phosphines 211
7. Isolation of Intact Mitochondria from Livers of Sprague-Dawley Rats 212
8. Chromatography of Soluble Mitochondrial Proteins 213
9. Labeling of Anion Exchange Eluates with 14-Azidotetradecanoyl-CoA and Phosphine-Biotin 213
10. Identification of Labeled Proteins by Mass Spectrometry 214
11. Demonstration that Acylation of Cysteine Residues Occurs via a Thioester Bond 215
12. Concluding Remarks 217
References 218
Section 2: Characterization of Mitochondrial Kinases, Phosphatases, Dynamics, and Respiratory Function 222
Chapter 10: Detection of a Mitochondrial Kinase Complex That Mediates PKA-MEK-ERK-Dependent Phosphorylation of Mitochondrial Proteins Involved in the Regulation of Steroid Biosynthesis 224
1. Introduction 225
2. Steroidogenic Cells 226
3. Analysis of a Mitochondrial Kinase Complex 227
4. Analysis of a Mitochondrial Acyl-CoA Thioesterase, Acot2 as a Phosphoprotein 241
References 245
Chapter 11: Isolation of Regulatory-Competent, Phosphorylated Cytochrome c Oxidase 248
1. Introduction 249
2. Purification of Mitochondria Maintaining Protein Phosphorylation 250
3. Isolation of Cytochrome c Oxidase from Mitochondria 254
4. Analysis of Cytochrome c Oxidase Phosphorylation 259
5. Concluding Remarks 263
References 264
Chapter 12: Using Functional Genomics to Study PINK1 and Metabolic Physiology 266
1. Introduction 267
2. Studying Mitochondrial Modulation in Humans In Vivo 269
3. Cellular Mitochondrial Models 271
4. Considerations When Using Short Interfering RNAs 273
5. Ex Vivo Gene Expression Analysis 277
References 281
Chapter 13: Functional Characterization of Phosphorylation Sites in Dynamin-Related Protein 1 286
1. Introduction 287
2. Replacement of Endogenous with Phosphorylation-Site Mutant Drp1 289
3. Analysis of Drp1 Phosphorylation 292
4. Production and Assays of Recombinant Drp1 295
5. Cell Death Assays 298
6. Mitochondrial Shape Analysis with ImageJ 300
7. Appendix: Morphometry Macro 305
References 306
Chapter 14: Functional Characterization of a Mitochondrial Ser/Thr Protein Phosphatase in Cell Death Regulation 310
1. Introduction 311
2. Identification of Protein Phosphatases in Mitochondria 313
3. Characterization of Mitochondrial Localization of PP2Cm in Mammalian Cells 315
4. Functional Characterization of PP2Cm in Cultured Cells 319
5. Functional Characterization of PP2Cm in Cell Death and Mitochondrial Regulation 322
6. Mitochondrial Phosphatase in Cell Death Regulation 325
Acknowledgments 326
References 326
Chapter 15: Distinguishing Mitochondrial Inner Membrane Orientation of Dual Specific Phosphatase 18 and 21 330
1. Introduction 331
2. Analysis of Phosphatase Activity of DSP18 332
3. Isolation of Highly Purified Rat Kidney Mitochondria and Subfractionation 334
4. Mitochondrial Inner Membrane Association of DSP18 and DSP21 338
Acknowledgments 341
References 342
Chapter 16: Monitoring Mitochondrial Dynamics with Photoactivateable Green Fluorescent Protein 344
1. Mitochondrial Dynamics 345
2. PAGFPmt 347
3. Tracking Individual Fusion and Fission Events 348
4. Quantifying Networking Activity in Whole Cells: Whole Cell Mitochondrial Dynamics Assay 351
5. Potential Artifacts and Important Controls 356
6. Comparison with an Alternative Method 357
Acknowledgments 358
References 358
Chapter 17: Determination of Yeast Mitochondrial KHE Activity, Osmotic Swelling and Mitophagy 360
1. Introduction 361
2. Disturbance of Mitochondrial K+ Homeostasis by dox-Regulated Mdm38 Expression 362
3. Morphological Changes in Mitochondria in Mdm38-Depleted Cells: Confocal Microscopy and Electron Microscopy 364
4. Measurement of KHE Activity and Other Bioenergetic Parameters 365
5. Following Mitophagy Using a Novel Biosensor and Fluorescence Microscopy 368
6. Growth and Preparation of Cells for Fluorescence Microscopy Observations of Mitophagy 369
7. Fluorescence Microscopy 370
Acknowledgments 371
References 372
Chapter 18: Imaging Axonal Transport of Mitochondria 374
1. Introduction 375
2. Protocols to Image Axonal Transport of Mitochondria in Rat Hippocampal Neurons 379
3. Protocols to Image Axonal Transport of Mitochondria in Drosophila Larval Neurons 383
4. Conclusion 386
Acknowledgments 387
References 387
Chapter 19: Generation of mtDNA-Exchanged Cybrids for Determination of the Effects of mtDNA Mutations on Tumor Phenotypes 390
1. Introduction 391
2. Establishment of rho0 Cells of Mice 392
3. Preparation and Enucleation of mtDNA-Donor Cells 395
4. Cell Fusion between rho0 Cells and Cytoplasts 396
5. Concluding Remarks 398
References 399
Section 3: Mitochondrial Function in Pancreatic Beta Cells and Insulin-Responsive Tissues 402
Chapter 20: Functional Assessment of Isolated Mitochondria In Vitro 404
1. Introduction 405
2. Mitochondrial Isolation Procedures 409
3. Mitochondrial ATP Production 415
4. Mitochondrial Respiration 420
5. Summary 424
References 424
Chapter 21: Assessment of In Vivo Mitochondrial Metabolism by Magnetic Resonance Spectroscopy 428
1. Introduction 429
2. In Vivo Magnetic Resonance Spectroscopy 430
3. Insulin Resistance and Resting Mitochondrial Metabolism 441
4. Conclusions 444
References 444
Chapter 22: Methods for Assessing and Modulating UCP2 Expression and Function 450
1. Introduction 451
2. Analysis of UCP2 Expression by Immunoblot 452
3. Analysis of UCP2 Expression by Real-Time PCR 454
4. Mitochondria Respiration Assay 455
5. Modulating UCP2 Expression by Cold Exposure 455
6. Inhibiting UCP2 Expression by Antisense Oligonucleotide 456
7. Inhibiting PGC-1alpha as an Indirect Means of Controlling UCP2 Expression 457
References 458
Chapter 23: Measuring Mitochondrial Bioenergetics in INS-1E Insulinoma Cells 460
1. Introduction 461
2. Quantification of Cellular Bioenergetics-Theoretical Aspects 462
3. INS-1E Cells-A Valuable Pancreatic Beta Cell Model 466
4. Measurement of Coupling Efficiency in Trypsinized INS-1E Cells 471
5. Noninvasive Measurement of Cellular Bioenergetics 473
Acknowledgment 477
References 478
Chapter 24: Investigating the Roles of Mitochondrial and Cytosolic Malic Enzyme in Insulin Secretion 480
1. Introduction 481
2. Malic Enzyme mRNA Expression in Rat Insulinoma INS-1 832/13 Cells, Rat Islets, and Mouse Islets 483
3. siRNA Knock-Down of ME1 and ME2 in INS-1 832/13 beta-Cells 487
4. Enzymatic Assays to Determine Activity of Cytosolic and Mitochondrial Malic Enzymes 490
5. Calculating Relative Rates of Anaplerotic Pathways from 13C-Glutamate Isotopomer Distribution 497
6. Discussion 503
References 504
Chapter 25: Insulin Secretion from beta-Cells is Affected by Deletion of Nicotinamide Nucleotide Transhydrogenase 506
1. Introduction 507
2. Gene-Driven ENU Screens 508
3. Generating BAC Transgenics 512
4. Isolation of Islets of Langerhans from Mouse Pancreas 515
5. RNA Interference (RNAi) Methodologies on Insulinoma Cell Lines 519
6. Measuring Intracellular Calcium 522
7. Measuring Hydrogen Peroxide in Mitochondria 523
8. Imaging of Mitochondrial Membrane Potential Changes by Confocal Microscopy 526
9. Measuring NNT Activity 530
10. Conclusions 532
References 533
Author Index 536
Subiect Index 564
Colour Plates 575