Visualizing Functional Flexibility by Three-Dimensional Electron Microscopy: Reconstructing Complex I of the Mitochondrial Respiratory Chain......Page 40
Introduction......Page 41
Random Conical Reconstruction......Page 43
Digitization......Page 44
Correction of the contrast transfer function......Page 45
Particle selection......Page 47
Processing of the 0deg images......Page 48
Refinement......Page 52
Sample preparation for electron microscopy......Page 53
2-D and 3-D data analysis......Page 54
References......Page 60
Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ......Page 65
Introduction......Page 66
Cell culture......Page 67
Confocal microscopy......Page 69
Dehydration, infiltration, and embedding......Page 70
Conventional TEM......Page 71
Electron microscope......Page 72
Computer and software......Page 74
Energy-filtering and serial tomography......Page 75
Estimation of tomographic resolution......Page 76
Size and number of fiducial gold particles......Page 77
Pre-irradiating ("cooking") the area of interest......Page 78
Angular increment and tilt range......Page 79
Microscope alignment, eucentricity, and focus......Page 80
Tilt series alignment......Page 81
Segmentation......Page 82
Movies......Page 83
Application to apoptotic HeLa cells......Page 84
References......Page 86
Studies of Complex I by Fourier Transform Infrared Spectroscopy......Page 89
Spectroscopic Studies of Complex I......Page 90
Analyses of complex I by FTIR difference spectroscopy......Page 92
Transmission FTIR studies......Page 93
ATR-FTIR studies......Page 94
Preparation of Rehydrated Layers of Complex I for ATR-FTIR Spectroscopy......Page 95
Analysis of Rehydrated Complex I Layers by Perfusion-Induced ATR-FTIR Difference Spectroscopy......Page 98
Model Compounds and Strategies for Interpretation......Page 100
Example IR Difference Spectra of Complex I......Page 105
References......Page 106
Electron Transfer in Respiratory Complexes Resolved by an Ultra-Fast Freeze-Quench Approach......Page 111
Comparison of the different flow techniques......Page 112
Ultra-Fast Freeze-Quench Setup......Page 115
Mixer design......Page 116
Fast freezing......Page 117
Sample Preparation......Page 118
Application of Freeze-Quench Approach for the Monitoring of Complex I Reduction by NADH......Page 121
References......Page 128
Use of Ruthenium Photooxidation Techniques to Study Electron Transfer in the Cytochrome bc1 Complex......Page 130
Introduction......Page 131
Design and Synthesis of Ruthenium-Labeled Proteins......Page 133
Method for the synthesis of ruthenium-labeled cytochrome c......Page 134
Measurement of Interprotein Electron Transfer in a Ruthenium-Labeled Protein......Page 135
Measurement of Electron Transfer between Ruz-39-Cc and Cytochrome bc1......Page 137
Measurement of Electron Transfer within the Cytochrome bc1 Complex......Page 140
References......Page 142
Mass Spectrometric Characterization of the Thirteen Subunits of Bovine Respiratory Complexes that are Encoded in Mitochondrial DNA......Page 145
Introduction......Page 146
The CMW extract of mitochondria......Page 149
Isolation of respiratory complexes......Page 151
CMW extraction of respiratory complexes......Page 152
HILIC fractionation of the PAHW extract of complex I......Page 153
Protein molecular mass measurements......Page 154
Tandem MS of protein ions......Page 157
Conclusions and Perspectives......Page 159
References......Page 162
Tracing Human Mitochondrial Complex I Assembly by Use of GFP-Tagged Subunits......Page 166
Introduction......Page 167
Prediction of steric hindrance by GFP......Page 169
Creation of Inducible Cell Line Expressing AcGFP1-Tagged NDUFS3......Page 170
Creation of the entry clone containing complex I subunit NDUFS3......Page 171
Creation of expression vector containing AcGFP1-tagged NDUFS3......Page 173
Stable transfection of expression vector containing AcGFP1-tagged NDUFS3......Page 174
Analysis of inducibility of stable cell line expressing AcGFP1-tagged NDUFS3......Page 175
Tracing AcGFP1-Labeled NDUFS3 on BN-PAGE......Page 176
Native gel analysis of complex I under leakage and induced condition......Page 177
Pulse-chase labeling of complex I and its assembly intermediates......Page 179
Accumulation of complex I assembly intermediates after chloramphenicol treatment......Page 181
References......Page 182
Two-Dimensional Native Electrophoresis for Fluorescent and Functional Assays of Mitochondrial Complexes......Page 185
Introduction......Page 186
Summary of electrophoretic and accessory protocols......Page 187
Gel and sample preparation......Page 188
2-D BN/SDS-PAGE......Page 190
2-D BN/hrCN electrophoresis......Page 191
2-D BN/SDS-PAGE......Page 193
2-D BN/hrCN electrophoresis......Page 196
Final 3-D SDS-PAGE or dSDS-PAGE......Page 198
References......Page 199
Reliable Assay for Measuring Complex I Activity in Human Blood Lymphocytes and Skin Fibroblasts......Page 201
Introduction......Page 202
Background......Page 203
Other solutions......Page 204
Isolation of blood lymphocytes......Page 205
Spectrophotometric assay......Page 206
Characteristics of the Complex I Assay......Page 207
References......Page 211
Purification of the Cytochrome c Reductase/Cytochrome c Oxidase Super Complex of Yeast Mitochondria......Page 214
Cultivation of yeast cells in lactate medium......Page 215
Separation of mitochondrial protein complexes by sucrose gradient ultracentrifugation......Page 216
Blue-native PAGE......Page 217
Perspectives......Page 218
References......Page 220
Supercomplex Organization of the Yeast Respiratory Chain Complexes and the ADP/ATP Carrier Proteins......Page 222
The OXPHOS complexes......Page 223
Supercomplex organization state of the OXPHOS system......Page 224
The supercomplex organization of yeast AAC proteins-the presence of AAC-cytochrome bc1-COX-TIM23 supercomplex......Page 226
Recrystallization of digitonin......Page 229
Isolation and Storage of Mitochondria......Page 230
BN-PAGE protein standard preparation......Page 231
Analysis after the SDS-PAGE and Western blotting steps......Page 232
Cloning and expression of HisAac2......Page 234
Affinity purification of His-tagged Aac2 and coassociating proteins......Page 236
References......Page 237
Controlled Expression of Iron-Sulfur Cluster Assembly Components for Respiratory Chain Complexes in Mammalian Cells......Page 240
Introduction......Page 241
Vector-based RNAi......Page 243
Electroporation-based transfections......Page 244
Assessing the efficiency of the RNAi treatment......Page 246
Assessing the specificity of the RNAi treatment......Page 247
Preparation and application of 55Fe-loaded transferrin......Page 249
Preparation of membranes containing respiratory chain complexes......Page 250
Blue-native PAGE and autoradiography......Page 251
Assessing the subunit composition of respiratory complexes by two-dimensional BN-PAGE......Page 252
Determination of lactate formation......Page 253
Determination of complex I activity by in-gel activity staining......Page 254
Analysis of enzyme activities in multiwell plates......Page 255
Determination of complex II (SDH) activity......Page 256
Preparing reduced cytochrome c......Page 257
Determination of citrate synthase activity......Page 258
References......Page 259
Localization and Function of the 2Fe-2S Outer Mitochondrial Membrane Protein mitoNEET......Page 263
Introduction......Page 264
Purification of Highly Enriched Mitochondria from Rat Liver......Page 265
Differential centrifugation to isolate crude mitochondria......Page 266
Histodenz gradient purification of mitochondria......Page 267
Fractionation of highly purified mitochondria......Page 269
Expression of Recombinant mitoNEET......Page 270
Generation of expression plasmids and mutagenesis......Page 271
Expression and purification of untagged mitoNEET......Page 272
Optical spectra of mitoNEET to assess redox status......Page 273
Assessing pH lability of cluster......Page 274
References......Page 275
Nucleotide-Dependent Iron-Sulfur Cluster Biogenesis of Endogenous and Imported Apoproteins in Isolated Intact Mitochondria......Page 277
Introduction......Page 278
Stock Solutions and Storage......Page 280
Isolation and Purification of Mitochondria......Page 281
Depletion of Endogenous Nucleotides in Isolated Mitochondria......Page 282
Insertion of newly formed [Fe-35S] clusters into endogenous apoaconitase and nucleotide (GTP and ATP) dependence......Page 283
Requirement for nucleotide hydrolysis......Page 285
Effects of matrix GTP levels......Page 287
[Fe-S] Cluster Biogenesis of Imported Apoferredoxin......Page 288
Bacterial expression and purification of unlabeled apoferredoxin precursor protein......Page 289
Insertion of newly formed [Fe-S] clusters into imported apoferredoxin-A single step coupled assay......Page 290
GTP-dependent [Fe-S] cluster biogenesis of imported apoferredoxin-A two-step assay......Page 291
Concluding Remarks and Perspectives......Page 293
Acknowledgments......Page 295
References......Page 296
Isolation of Saccharomyces Cerevisiae Mitochondria for Moumlssbauer, Epr, and Electronic Absorption Spectroscopic Analyses......Page 297
Introduction......Page 298
Large-Scale Growth of S. Cerevisiae......Page 300
Anaerobic Isolation......Page 302
Isolation Procedure......Page 303
Determining The Absolute [Fe] and [Protein] in Mitochondria......Page 305
Packing Samples into Spectroscopy Holders......Page 307
Advances with This Approach......Page 311
Acknowledgments......Page 313
References......Page 314
The Use of Fluorescence Correlation Spectroscopy to Probe Mitochondrial Mobility and Intramatrix Protein Diffusion......Page 316
Introduction......Page 317
Principle of Fluorescence Correlation Spectroscopy......Page 318
Calibration of the FCS setup......Page 321
Data analysis......Page 323
Data interpretation......Page 326
References......Page 328
Type II NADH: Quinone Oxidoreductases of Plasmodium Falciparum and Mycobacterium Tuberculosis: Kinetic and High-Throughput Assays......Page 332
Structure and function......Page 333
Functional role of ndh in the respiratory chain of P. falciparum and M. tuberculosis......Page 334
PfNDH2 native assay......Page 337
PfNDH2 linked assay......Page 338
Production of crude membrane preparations from E. coli F571......Page 340
NADH: Ubiquinone oxidoreductase assay......Page 341
Endpoint Assay for HTS......Page 344
Acknowledgments......Page 347
References......Page 348
Analysis of Respiratory Chain Complex Assembly with Radiolabeled Nuclear- and Mitochondrial-Encoded Subunits......Page 350
Introducton......Page 351
Pulse-chase labeling of mtDNA-encoded translation products in cultured cells......Page 353
Sample preparation......Page 354
Mitochondrial Import and Assembly of Nuclear DNA-Encoded Subunits......Page 355
In vitro transcription......Page 356
Coupled in vitro transcription/translation......Page 357
Mitochondrial in vitro import and assembly assay......Page 358
Chase assays......Page 359
Separation of Protein Complexes with Blue-Native PAGE......Page 360
Sample preparation and electrophoresis conditions......Page 361
Separation of Individual Subunits from their Complexes with BN-PAGE and SDS-PAGE in the Second Dimension......Page 362
Analysis......Page 363
Acknowledgments......Page 366
References......Page 367
Measuring Redox Changes to Mitochondrial Protein Thiols With Redox Difference Gel Electrophoresis (Redox-Dige)......Page 369
Introduction......Page 370
Overview of Redox-Dige......Page 371
Experimental Design......Page 373
Reduction of redox-modified thiols......Page 374
Labeling of redox-modified thiols with fluorescent maleimides......Page 375
Imaging of Fluorescently Labeled Protein on Gels......Page 377
Analysis of Multiple Gels to Determine Statistically Significance of Redox Changes on Individual Proteins......Page 379
Spot Excision From 2-D Gels......Page 380
Protein Spot Identification by Mass Spectrometry......Page 381
Limitations to Redox-Dige and Future Possible Adaptations......Page 382
Comparison of Redox-Dige With Other "Redox Proteomic" Techniques......Page 383
References......Page 384
Effects of Hepatitis C Core Protein on Mitochondrial Electron Transport and Production of Reactive Oxygen Species......Page 388
Introduction......Page 389
Materials......Page 390
Plasmid design and core protein isolation......Page 391
Cellular model systems......Page 393
Effects of core protein on mitochondrial respiration......Page 394
Direct effect of core on mitochondrial GSH/GSSG......Page 395
Measurement of core effects on calcium uptake in isolated mitochondria......Page 396
Mitochondrial Effects of Viral Proteins in Cellular Systems......Page 398
Measurement of core protein effects on live cell mitochondrial Ca2+......Page 399
Core protein effects on mitochondrial ROS production......Page 402
Acknowledgments......Page 403
References......Page 404
Paraquat-Induced Production of Reactive Oxygen Species in Brain Mitochondria......Page 406
Introduction......Page 407
Isolation of rat brain mitochondria......Page 408
Polarographic measurement......Page 409
Procedure......Page 411
Fluorometric measurement......Page 412
Discussion......Page 413
References......Page 416
The Uptake and Interactions of the Redox Cycler Paraquat with Mitochondria......Page 419
Paraquat (PQ) as a redox cycler......Page 420
PQ and mitochondria......Page 421
PQ and Parkinson's disease......Page 422
Detection of the PQ+ radical by EPR......Page 423
Detection of the PQ+ radical by spectrophotometry......Page 424
Radioactive [14C]-PQ......Page 425
Construction of a PQ-selective electrode......Page 426
Aconitase inacti
vation......Page 428
Coelenterazine (CLZ) chemiluminescence......Page 431
Amplex Red fluorescence......Page 432
Measurement of PQ uptake with an ion-selective electrode......Page 433
Measurement of PQ uptake by EPR......Page 434
Measurement of PQ uptake from the distribution of radiolabeled [14C]-PQ......Page 436
PQ Toxicity Screens in Yeast Deletion Libraries......Page 437
Conclusions......Page 438
References......Page 439
Quantification of Superoxide Production by Mouse Brain and Skeletal Muscle Mitochondria......Page 442
Fluorimetric determination of hydrogen peroxide by peroxidase mediated oxidation of p-hydroxyphenylacetate (pHPAA)
......Page 443
Fluorimetric determination of hydrogen peroxide by peroxidase-mediated oxidation of Amplex red to resorufin......Page 444
Determination of superoxide production
......Page 446
Fluorimetric detection of superoxide production with oxidation of hydroethidine
......Page 448
Photometric detection of superoxide by reduction of acetylated cytochrome c......Page 450
The Application of Quantitative Methods for Measurement of ROS Production to Identify the Contribution of Individual Sites to the Superoxide Production of Isolated Brain and Skeletal Muscle Mitochondria......Page 451
Brain and skeletal muscle preparations used in the study......Page 452
Quantification of superoxide production of rat brain SMP......Page 453
Relationship between maximal respiration and complex I-related ROS generation of different brain subcellular preparations
......Page 454
Tissue dependency of mitochondrial superoxide generation rates-comparison of mouse brain and skeletal muscle mitochondria......Page 455
References......Page 459
Quantification, Localization, and Tissue Specificities of Mouse Mitochondrial Reactive Oxygen Species Production......Page 461
Introduction......Page 462
Procedure for the isolation of mouse brain mitochondria......Page 464
Liver mitochondria......Page 465
Submitochondrial particles (SMPs)......Page 466
Procedure for the production of SMPs......Page 467
ROS production by intact mitochondria......Page 468
ROS production by alamethicin-permeabilized mitochondria......Page 469
Reagents......Page 470
ROS production by SMP......Page 471
Reagents and Procedures for Evaluating Ros Production by Beta Cells......Page 472
Reagents......Page 473
Reagents......Page 474
Acknowledgments......Page 475
References......Page 476
Analysis of Electron Transfer and Superoxide Generation in the Cytochrome bc1 Complex......Page 480
Introduction......Page 481
Materials......Page 482
Preparation of submitochondrial particles (SMP) from frozen bovine heart muscles......Page 483
Preparation of succinate: cytochrome c oxidoreductase from SMP......Page 484
Preparation of cyt bc1 complex from cyt bc1 particles......Page 485
Electron Transfer Activity in the Purified bc1 Complex......Page 486
Proton Translocation in the Purified bc1 Complex......Page 487
Superoxide Generation by the Purified bc1 Complex......Page 488
Comparison of O.2- Production by the bc1 Complexes with Varying Electron Transfer Activities......Page 489
Acknowledgments......Page 491
References......Page 492
Measurement of Superoxide Formation by Mitochondrial Complex I of Yarrowia Lipolytica......Page 495
Introduction......Page 496
Materials......Page 497
Purification of complex I, lipid-activation, and reconstitution into proteoliposomes......Page 498
Detection of Superoxide (O.2-) by Acetylated Cytochrome c......Page 499
Superoxide production by mitochondrial membranes of Y. lipolytica......Page 500
Superoxide production by reconstituted complex I......Page 501
pH profile for ubiquinone reduction and superoxide formation......Page 503
Parallel measurement of NADH oxidation, H2O2 generation, and buildup of a membrane potential by reconstituted complex I......Page 504
Conclusions......Page 507
References......Page 508
An Improved Method for Introducing Point Mutations into the Mitochondrial Cytochrome b Gene to Facilitate Studying the Role of Cytochrome b in the Formation of Reactive Oxygen Species......Page 511
Introduction: The Role of Cytochrome b in Superoxide Anion Formation......Page 512
Construction of Plasmid pSCSI Containing an ARG8m Cassette Flanked by the Cytochrome b Gene 5prime - and 3prime -UTR Sequences......Page 514
Construction of the Yeast Recipient Strain for Cytochrome b Mutations, YTE31, In which the Cytochrome b Gene is Replaced by Mitochondrially Encoded ARG8......Page 515
Construction of the Template Vector, Plasmid pMD2, Containing an Intronless Cytochrome b Gene and COX2......Page 517
Creation of Yeast Cytochrome b Mutant Strains by Biolistic Transformation of Yeast Strain DFS160 rhoo with Plasmids Containing Mutated Versions of the Cytochrome b Gene......Page 518
Obtaining cytochrome b respiratory-competent mutations in a rho+ background......Page 520
Cytoduction between Recipient Strain YTE31 and MR6, A Derivative of W303-1B......Page 522
Creating Recipient Strains FG20 and FG21, Derivatives of W303-1B that Contain the YTE31 Mitochondria......Page 523
References......Page 525
Use of Ruthenium Photoreduction Techniques to Study Electron Transfer in Cytochrome Oxidase......Page 527
Introduction......Page 528
Materials......Page 529
Preparation of ruthenium-labeled cytochrome c......Page 530
Measurement of Intraprotein Electron Transfer in Horse Ru-39-Cc......Page 531
Measurement of Electron Transfer from RuD-39-Cc to Cytochrome c Oxidase......Page 533
Measurement of formation and dissociation rate constants for the complex between Ru-Cc and CcO......Page 534
Measurement of Electron Transfer and Oxygen Reduction in Cytochrome c Oxidase with Electrostatically Bound Ruthenium Complexes......Page 535
Measurement of single electron reduction of CcO state Pm to F......Page 536
Measurement of single electron reduction of F state to O state......Page 537
Measurement of single electron reduction of state E to states R2 and Pm......Page 538
References......Page 539