Mitochondrial Function: Mitochondrial Protein Kinases, Protein Phosphatases and Mitochondrial Diseases Part B

Cover Page
......Page 1
Series Editor
......Page 2
Series Editor......Page 0
Copyright Page......Page 3
Contributors......Page 4
Preface......Page 13
Methods in Enzymology......Page 15
The Mitochondrial Proteome Database: MitoP2......Page 42
Introduction......Page 43
Yeast, Human, and Mouse Proteome Information......Page 45
Mitochondrial Reference Set, Candidates, and SVM Prediction Score......Page 47
Proteome, transcriptome, and sub-localization experiments in yeast......Page 48
Proteome, transcriptome, and sub-localization experiments in human and mouse......Page 49
Other Search Options: Restrictions to Functional Categories, Gene Locus and Disease-Causing Proteins......Page 51
Output Lists and Practical Examples......Page 53
References......Page 56
Predicting Proteomes of Mitochondria and Related Organelles from Genomic and Expressed Sequence Tag Data......Page 60
Introduction......Page 61
Isolation of synaptic mouse brain mitochondria......Page 62
Mitosomes......Page 63
Organellar proteome characterization......Page 64
Mitochondrial targeting and import......Page 65
Computational methods for predicting mitochondrial localization......Page 66
Data Preparation......Page 67
CBOrg......Page 68
MITOPRED......Page 71
Cell Fusion between rho0 Cells and Cytoplasts......Page 72
TargetP......Page 73
Sequence feature-based prediction for prediction of mitochondrial localization......Page 74
CELLO......Page 75
PSORT......Page 76
SherLoc......Page 77
YimLOC......Page 78
Comparison of Methods......Page 80
Conclusion......Page 82
References......Page 83
Proteome Characterization of Mouse Brain Mitochondria Using Electrospray Ionization Tandem Mass Spectrometry......Page 87
Mitochondrial Dynamics......Page 88
Analysis of Mitochondrial Proteome by Two-Dimensional PAGE and LC-MS Techniques......Page 89
Insulin Resistance and Resting Mitochondrial Metabolism......Page 411
Analysis of Mouse Brain Mitochondria Using the CITP-Based Proteome Platform......Page 90
Coupling transient CITP/CZE-based multidimensional separations with electrospray ionization (ESI)-MS......Page 91
Monitoring metabolic fluxes using 13C MRS......Page 405
Proteome Characterization of Mouse Brain Mitochondria......Page 93
References......Page 98
32P Labeling of Protein Phosphorylation and Metabolite Association in the Mitochondria Matrix......Page 101
Introduction......Page 102
Isolation of Mitochondria and Mitochondrial Membranes......Page 137
Mitochondria isolation and phosphate loading......Page 104
32P labeling of intact mitochondria......Page 106
Generation of probe......Page 178
Mass spectrometry......Page 109
Results and Discussion......Page 110
Running the experiment and quantitation of ATP......Page 387
Isolation of CPT-I......Page 111
Chase experiments with cold phosphate......Page 112
Suppression of PDH......Page 113
Confirmation of siRNA specific knockdown......Page 489
Blue native gel electrophoresis......Page 115
Summary......Page 116
References......Page 117
Selective Enrichment in Phosphopeptides for the Identification of Phosphorylated Mitochondrial Proteins......Page 119
Introduction......Page 120
Immobilized metal affinity chromatography (IMAC)......Page 122
Verification of novel transcripts......Page 258
Single-tube and microtip methods using metal oxides......Page 123
Single-tube experiment applying bare metal oxide particles......Page 124
Chromatography of mitochondrial extracts......Page 159
Single-tube experiment applying Zr4+- and Ti4+-IMAC beads......Page 125
Methyl esterification to increase selectivity......Page 126
Precursor ion scanning......Page 127
On-Line 2D-LC Phosphopeptide Enrichment Methods......Page 128
2D-SCX-TiO2-RP-LC-MS......Page 129
Mass Spectrometry-Based Methods......Page 130
Insulin secretion experiment from static incubation......Page 487
ESI-MS and data dependent MS/MS strategies......Page 131
References......Page 132
Post-translational Modifications of Mitochondrial Outer Membrane Proteins......Page 135
Introduction......Page 136
Preparation of mitochondrial outer membranes......Page 138
Detection of Post-translational Modifications Using One-dimensional Gel Electrophoresis......Page 139
Reduction, alkylation, and enzymatic digestion of the protein......Page 141
Detection of post-translational modifications using mass spectrometry......Page 142
Antibody preparation and immobilization......Page 143
Mass spectrometric analysis of the isolated CPT-I......Page 145
Shotgun Methods for Identification of Post-translational Modifications......Page 147
Mass spectrometric analysis of digestion......Page 148
Acknowledgments......Page 149
References......Page 150
Analysis of Tyrosine-Phosphorylated Proteins in Rat Brain Mitochondria......Page 154
Introduction......Page 155
Quantification of Cellular Bioenergetics-Theoretical Aspects......Page 431
Purification of Src family tyrosine kinase Lyn and Fgr......Page 156
Isotopic mass balance equations......Page 157
Assay of tyrosine kinase activity......Page 158
Tyrosine phosphorylation of mitochondrial proteins......Page 160
Specific immunoaffinity-based enrichment of pY-containing peptides......Page 162
General enrichment of phosphopeptides by TiO2-affinity chromatography......Page 164
Common mass spectrometric identification......Page 165
Multiple reaction monitoring......Page 167
Bioinformatic Tools to Analyze the Potential Role of Tyrosine Phosphorylation......Page 168
References......Page 171
Acetylation of Mitochondrial Proteins......Page 174
Purification of Enzymatically Active SIRT3......Page 175
Purification of SIRT3 from mammalian cell culture systems......Page 176
Purification of SIRT3 from bacterial expression systems......Page 177
Studies in human muscle primary myotubes......Page 249
Enzymatic assay......Page 179
Immunoprecipitation and immunoblotting......Page 180
Spatially precise photoconversion with 2-photon laser......Page 181
Detection of acetylated mitochondrial proteins by immunoprecipitation......Page 182
Concluding Remarks......Page 183
References......Page 184
Non-radioactive
Detection of Palmitoylated Mitochondrial Proteins Using an Azido-Palmitate Analogue......Page 185
Introduction......Page 186
Methyl 12-hydroxydodecanoate......Page 189
Immunoblotting of UCP2 in samples from hypothalamus and pancreatic islet mitochondria......Page 190
siRNA Knock-Down of ME1 and ME2 in INS-1 832/13 beta-Cells......Page 191
Protein acylation reaction......Page 192
Isolation of Intact Mitochondria from Livers of Sprague-Dawley Rats......Page 193
Labeling of Anion Exchange Eluates with 14-Azidotetradecanoyl-CoA and Phosphine-Biotin......Page 194
Mass spectrometric analysis of peptide extracts and database searching......Page 195
Demonstration that Acylation of Cysteine Residues Occurs via a Thioester Bond......Page 196
In-gel neutral hydroxylamine treatment......Page 197
Concluding Remarks......Page 198
References......Page 199

Detection of a Mitochondrial Kinase Complex That Mediates PKA-MEK-ERK-Dependent Phosphorylat......Page 202
Introduction......Page 203
Steroidogenic Cells......Page 204
Involvement of PKA, MEK1/2, and ERK1/2 in mitochondrial function......Page 205
Constructs of wild-type ERK and ERK H230R for transfection and expression in MA-10 cells......Page 206
Subcellular fractionation and assessment of the purity of each fraction......Page 207
Western blot analysis of ERK1/2 and MEK1/2......Page 208
Confocal analysis of ERK1/2......Page 209
StAR construct and MA-10 cell transfection to enrich the mitochondria of non-stimulated cells with StAR......Page 211
Cell-free assay as a tool to evaluate mitochondrial function......Page 212
Interaction of StAR with the kinase complex: Pull-down assay......Page 214
Mutation of the putative ERK1/2 phosphorylation site: Obtention of recombinant wild type and S232A mutated StAR protein......Page 215
In vitro phosphorylation of recombinant wild type and S232A StAR by wild type and inactive ERK1......Page 216
Analysis of Ser232 as the phosphorylation site in phospho-StAR: Limited digestion by V8 endoprotease and separation......Page 217
Effect of StAR phosphorylation by ERK1 on residue Ser232 on StAR cellular function......Page 218
Analysis of a Mitochondrial Acyl-CoA Thioesterase, Acot2 as a Phosphoprotein......Page 219
Purification of Acot2 to homogeneity preserving its biological activity......Page 220
Identification of Acot2 as a phosphoprotein......Page 221
References......Page 223

Isolation of Regulatory-Competent, Phosphorylated Cytochrome......Page 226
Introduction......Page 227
Purification of Mitochondria Maintaining Protein Phosphorylation......Page 228
General considerations......Page 229
Incubation of ground tissue with compounds that modify signaling pathways (optional)......Page 230
Isolation of mitochondria......Page 231
Determination of cytochrome c oxidase concentration and purity......Page 232
Cytochrome c oxidase isolation......Page 234
Analysis of Cytochrome c Oxidase Phosphorylation......Page 237
An oxygraph protocol for mitochondrial assessment......Page 238
Phosphoepitope determination via immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ioni......Page 239
Concluding Remarks......Page 241
References......Page 242

Using Functional Genomics to Study PINK1 and Metabolic Physiology......Page 244
Introduction......Page 245
Increase in mitochondrial activity......Page 247
Mitochondrial dysfunction......Page 248
Knockdown of target genes in brown adipocytes......Page 250
Considerations When Using Short Interfering RNAs......Page 251
Off-target effects of siRNA......Page 252
siRNA and apoptosis......Page 253
RNA isolation......Page 255
Global gene expression analysis-microarray......Page 256
Targeted gene expression-quantitative real-time PCR......Page 257
References......Page 259

Functional Characterization of Phosphorylation Sites in Dynamin-Related Protein 1......Page 263
Introduction......Page 264
Replacement of Endogenous with Phosphorylation-Site Mutant Drp1......Page 266
RNA isolation......Page 267
Site-directed mutagenesis......Page 268
Analysis of Drp1 Phosphorylation......Page 269
Metabolic labeling with 32P and immunoprecipitation of Drp1......Page 270
Generation of phosphorylation-specific antibodies......Page 271
Production and Assays of Recombinant Drp1......Page 272
Expression and purification of recombinant Drp1......Page 273
GTPase activity assays......Page 274
Viability assays in stable PC12 cell lines......Page 275
Apoptosis/necrosis assays in primary astrocytes......Page 276
Mitochondrial Shape Analysis with ImageJ......Page 277
Image enhancement......Page 278
Morphometry......Page 281
Inhibiting PGC-1alpha as an Indirect Means of Controlling UCP2 Expression......Page 282
References......Page 283
Functional Characterization of a Mitochondrial Ser/Thr Protein Phosphatase in Cell Death Regulation......Page 286
Introduction......Page 287
Molecular cloning of mitochondria PP2Cm......Page 289
Detecting mitochondrial localization of PP2Cm by immunofluorescent microscopy......Page 291
Determination of mitochondrial targeting of PP2Cm via fractionation......Page 292
Osmotic shock assay......Page 293
Colorimetric assay......Page 295
Knocking down PP2Cm with siRNA expressing vector......Page 296
Adenovirus-mediated gene deliver in mouse liver......Page 297
PP2Cm deficiency on cell viability and mitochondria inner membrane potential using JC-1 fluorescent assay......Page 298
PP2Cm deficiency on mitochondria oxidative phosphorylation and respiration......Page 299
Mitochondrial Phosphatase in Cell Death Regulation......Page 301
References......Page 302
Distinguishing Mitochondrial Inner Membrane Orientation of Dual Specific Phosphatase 18 and 21......Page 305
Introduction......Page 306
Bacterial expression constructs and mutagenesis......Page 307
Expression and purification of recombinant DSP18......Page 308
Isolation of Highly Purified Rat Kidney Mitochondria and Subfractionation......Page 309
Differential centrifugation to isolate crude mitochondria......Page 310
Histodenz gradient purification of mitochondria......Page 311
Subfractionation of highly purified mitochondria......Page 312
Trypsin digestions of mitochondrial inner membranes......Page 313
Integral versus peripheral membrane protein......Page 315
Acknowledgments......Page 316
References......Page 317
Monitoring Mitochondrial Dynamics with Photoactivateable Green Fluorescent Protein......Page 318
Monitoring dynamics......Page 320
Targeting mitochondrial matrix......Page 321
Reverse transcription of RNA to cDNA......Page 453
Tracking Individual Fusion and Fission Events......Page 322
Co-labeling mitochondria with TMRE versus dsRED versus Mitotracker Red......Page 323
Quantifying Networking Activity in Whole Cells: Whole Cell Mitochondrial Dynamics Assay......Page 325
Photoconversion of mitochondrial subpopulations for the whole cell mitochondrial dynamics assay......Page 326
Time lapse confocal imaging for cellular mitochondrial dynamics quantification......Page 327
Monitoring GFP dilution......Page 328
Monitoring GFP spread......Page 329
Photobleaching and saturation......Page 330
Comparison with an Alternative Method......Page 331
Acknowledgments......Page 332
Determination of Yeast Mitochondrial KHE Activity, Osmotic Swelling and Mitophagy......Page 334
Disturbance of Mitochondrial K+ Homeostasis by dox-Regulated Mdm38 Expression......Page 336
Morphological Changes in Mitochondria in Mdm38-Depleted Cells: Confocal Microscopy and Electron Microscopy......Page 338
KOAc-induced swelling......Page 339
KHE measurements with fluorescent dyes entrapped in submitochondrial particles......Page 340
Membrane potential......Page 341
The novel biosensor......Page 342
Transformation of yeast cells with biosensor constructs......Page 343
Fluorescence Microscopy......Page 344
Acknowledgments......Page 345
References......Page 346
Imaging Axonal Transport of Mitochondria......Page 347
Introduction......Page 348
Imaging mitochondrial movement in Drosophila larval axons......Page 350
Primary neuronal culture......Page 352
Image analysis......Page 353
Expected results......Page 354
13C NMR analysis of the 13C isotopic isomers of glutamate......Page 356
Image analysis......Page 357
Expected results......Page 358
Conclusion......Page 359
References......Page 360
Generation of mtDNA-Exchanged Cybrids for Determination of the Effects of mtDNA Mutations on Tumor Phenotype......Page 362
Introduction......Page 363
Establishment of rho0 Cells of Mice......Page 364
General tissue culture procedures......Page 435
Picking up the colonies and confirmation of their lack of mtDNA......Page 365
Preparation of mtDNA-donor cells......Page 367
Enucleation of mtDNA-donor cells......Page 368
Selection......Page 369
References......Page 502
Functional Assessment of Isolated Mitochondria in Vitro......Page 374
Introduction......Page 375
Mitochondrial Isolation Procedures......Page 379
Tissue homogenization......Page 380
Separation of mitochondria by differential centrifugation......Page 382
Mitochondrial yield and integrity......Page 383
Principles of the bioluminescent approach......Page 385
Preparation of substrates and ADP......Page 386
Overview......Page 390
Summary......Page 394
References......Page 447
Assessment of in Vivo Mitochondrial Metabolism by Magnetic Resonance Spectroscopy......Page 398
Introduction......Page 399
In Vivo Magnetic Resonance Spectroscopy......Page 400
High-resolution DNA melting analysis......Page 402
Measuring muscle ATP synthesis in vivo by 31P saturation-transfer MRS......Page 404
Metabolic modeling of the TCA cycle flux......Page 409
Isotope balance......Page 410
Resting mitochondrial metabolism versus maximal capacity......Page 413
References......Page 414
Two Methods for Assessing and Modulating UCP2 Expression and Function......Page 419
Introduction......Page 420
Isolating pancreatic islets......Page 421
Preparing mitochondria from hypothalamus and pancreatic islets......Page 422
Real-time PCR......Page 423
Modulating UCP2 Expression by Cold Exposure......Page 424
Measuring Intracellular Calcium......Page 491
References......Page 427
Measuring Mitochondrial Bioenergetics in INS-1E Insulinoma Cells......Page 429
Introduction......Page 430
Preparation of mitochondrial samples......Page 437
Western analysis......Page 438
UCP2 knockdown by RNAi......Page 439
Measurement of Coupling Efficiency in Trypsinized INS-1E Cells......Page 440
Noninvasive Measurement of Cellular Bioenergetics......Page 442
Measurement of coupling efficiency in attached INS-1E cells-Probing the effect of UCP2 knockdown......Page 443
Modular-kinetic measurement of INS-1E bioenergetics-Practical considerations for future experiments......Page 444
Acknowledgment......Page 446
Investigating the Roles of Mitochondrial and Cytosolic Malic Enzyme in Insulin Secretion......Page 449
Introduction......Page 450
Malic Enzyme mRNA Expression in Rat Insulinoma INS-1 832/13 Cells, Rat Islets, and Mouse Islets......Page 452
RTqPCR for ME1 and ME2 mRNA expression in cells and islets......Page 454
ME1 and ME2 siRNA design......Page 457
Protocol for experimental siRNA KD of ME1 and ME2 in INS-1 832/13 beta-cells......Page 458
Cytosolic malic enzyme activity assay......Page 459
Malic Enzyme 1 assay reaction mixture stock components......Page 460
Mitochondrial malic enzyme activity assay......Page 461
Isolation of mitochondria......Page 462
Cytochrome c assay......Page 463
Kinetic analysis of mitochondrial malic enzyme activity......Page 464
Electrospray tandem mass spectrometry for determining 13C-enrichment of Kreb's cycle intermediates and pyruvate......Page 465
Calculating Relative Rates of Anaplerotic Pathways from 13C-Glutamate Isotopomer Distribution......Page 466
13C-labeling of cells for isotopomer studies of anaplerotic pathways......Page 468
Analysis of glutamate 4......Page 469
Calculation of metabolic fluxes with tcacalc......Page 470
Discussion......Page 472
References......Page 473
Insulin Secretion from beta-Cells is Affected by Deletion of Nicotinamide Nucleotide Transhydrogenase......Page 475
Denaturing high-performance liquid chromatography (DHPLC)......Page 477
Rederivation of live mice......Page 480
Hybridization......Page 481
BAC transgenesis......Page 482
Isolation of Islets of Langerhans from Mouse Pancreas......Page 484
Isolation methods for obtaining islets......Page 485
Gene silencing using siRNA......Page 488
Measuring Hydrogen Peroxide in Mitochondria......Page 492
Imaging of Mitochondrial Membrane Potential Changes by Confocal Microscopy......Page 495
Confocal imaging of mitochondrial potential in pancreatic beta-cells......Page 496
Analysis of confocal images representing the mitochondrial membrane potential......Page 498
Microassay of NNT......Page 499
Conclusions......Page 501
Author Index......Page 505
Subject Index......Page 532