Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

Summary

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

Abstract

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

Introduction

Mitochondrial energy in the form of ATP is not only essential for cell survival, but also for the regulation of cell death. The generation of ATP by oxidative phosphorylation requires a functional electron transport chain (ETC; Cx-I to IV) and mitochondrial ATP synthase (Cx-V). Recent studies have shown that these large protein complexes are organized into supercomplexes, called respirasomes and synthasomes^1,2. It is challenging to analyze the assembly, dynamics, and activity regulation of these massive complexes and supercomplexes. While oxygen consumption measurements taken with an oxygen electrode and enzym....

Protocol

All experiments were performed using hearts from C57BL/6N mice (wild type). Mice were anesthetized with CO₂ prior to cervical dislocation, and all procedures were performed in strict accordance with the Division of Laboratory Animal Medicine at the University of Rochester and in compliance with state law, federal statute, and NIH policy. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Rochester (University Committee on Animal Resources).

1. CN and BN PAGE

NOTE: All equipment used for BN and CN PAGE must be free of detergent. To ensure this, wash all equip....

Results

To visualize mitochondrial supercomplexes, freshly isolated mitochondria from mice were used^17,18. Mitochondrial supercomplexes are sensitive to repeated cycles of freezing and thawing, leading to their disintegration, although this may be tolerable for some researchers. If freezing is necessary for storage, to ensure best results, samples should not undergo more than one cycle of freezing and thawing.

Discussion

A functional ETC is necessary for mitochondrial ATP generation. The complexes of the ETC are able to form two types of supercomplexes: the respirasomes (Cx-I, -III, and -IV)¹ and the synthasomes (Cx-V)². The assembly of each complex is required for an intact ETC, while the organization of the ETC into supercomplexes is thought to increase overall ETC efficiency^5,22. How these supercomplexes assemble and disassemble .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protean II mini-gel chamber	Biorad	1658004	Complete set to pour and run mini-gel electrophoresis
Protean XL maxi-gel	Biorad	1653189	Complete set to pour and run maxi-gel electrophoresis
Gradient maker, Hoefer SG15	VWR	95044-704	Pouring mini-gel gradients
Gradient maker, maxi-gel	VWR	GM-100	Pouring maxi-gel gradients
Transfer kit	Biorad	1703930	Complete set to wet transfer of proteins onto membranes
Electroeluter model 422	Biorad	1652976	Electroelution of proteins from native or SDS PAGES
Glass plates	Biorad	1653308	Short plates
Glass plates	Biorad	1653312	Spacer plates
Glass plates	Biorad	1651823	Inner plates
Glass plates	Biorad	1651824	Outer Plates
Power supply	Biorad	1645070	Power supply suitable for native electrophoresis
ECL-Western	Thermo Scientific	32209	Chemolumniscense substrate
SuperSignal-West Dura	Thermo Scientific	34075	Enhanced chemolumniscense substrate
Film/autoradiography film	GE Health care	28906845	Documentation of Western blots
Film processor CP1000	Agfa	NC0872640
Canon Power Shot 640	Canon	NA	Taking photos to document gels, membranes and blots.
Canon Power Shot 640 Camera hood	Canon		shielding camera for photos being taken on a light table
Acrylamide/bisacrylamide	Biorad	1610148	40% pre-mixed solution
Glycine	Sigma	G7403
SDS (sodium dodecyl sulfate)	Invitrogen	15525-017
Tris-base	Sigma	T1503	Buffer
Tricine	Sigma	T0377
Sodium deoxychelate	Sigma	D66750	Detergent
EDTA	Sigma	E5134
Sucrose	Sigma	S9378
MOPS	Sigma	M1254	Buffer
Imidazole	Sigma	I15513	Buffer
Lauryl maltoside	Sigma	D4641	Detergent
Coomassie G250	Biorad	161-0406
Aminohexanoic acid	Sigma	O7260
Native  molecular weight kit	GE Health care	17-0445-01	High molecular weight calibraition kit for native electrophoresis.
Name	Company	Catalog Number	Comments
NADH	Sigma	N4505
Nitroblue tetrazolium	Sigma	N6639
Tris HCl	Sigma	T3253
ATP	Sigma	A2383
Name	Company	Catalog Number	Comments
Lead(II) nitrate (Pb(NO3)2):	Sigma	228621
Oligomycin	Sigma	O4876
Name	Company	Catalog Number	Comments
Ponceau S	Sigma	P3504
anti-ATP5A	Abcam	ab14748	antibody to ATP synthase subunit ATP5A
anti-NDUFB6	Abcam	ab110244	antibody to Cx-1 subunit NDUFB6
anti-VDAC	Calbiochem	529534	antibody to VDAC
ECL HRP linked antibody	GE Health Care	NA931V	secondary antibody to ATP5A, NDUFB6 and VDAC
Blocking reagent	Biorad	170-6404
BSA
sodium chloride	Sigma	S9888
potassium chloride	Sigma	P9541
EGTA	Sigma	E3889
Name	Company	Catalog Number	Comments
Silver staining Kit	Invitrogen	LC6070