Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis

doi:10.1016/j.ab.2007.02.022

Analytical Biochemistry

Volume 364, Issue 2, 15 May 2007, Pages 128-137

https://doi.org/10.1016/j.ab.2007.02.022 Get rights and content

Abstract

The mitochondrial respiratory chain consists of multi-subunit protein complexes embedded in the inner membrane. Although the majority of subunits are encoded by nuclear genes and are imported into mitochondria, 13 subunits in humans are encoded by mitochondrial DNA. The coordinated assembly of subunits encoded from two genomes is a poorly understood process, with assembly pathway defects being a major determinant in mitochondrial disease. In this study, we monitored the assembly of human respiratory complexes using radiolabeled, mitochondrially encoded subunits in conjunction with Blue Native polyacrylamide gel electrophoresis. The efficiency of assembly was found to differ markedly between complexes, and intermediate complexes containing newly synthesized mitochondrial DNA-encoded subunits could be observed for complexes I, III, and IV. In particular, we detected human cytochrome b as a monomer and as a component of a novel approximately 120 kDa intermediate complex at early chase times before being totally assembled into mature complex III. Furthermore, we show that this approach is highly suited for the rapid detection of respiratory complex assembly defects in fibroblasts from patients with mitochondrial disease and, thus, has potential diagnostic applications.

Section snippets

Cell lines and culture conditions

143BTK⁻ ρ⁺ (wild-type) and 143B-87 ρ⁰ (lacking mtDNA) osteosarcoma cells were a gift from I. Trounce (University of Melbourne). Fibroblast cell lines were derived from skin biopsies of control (wild-type) or mitochondrial disease patients with lethal infantile mitochondrial disease (LIMD) (patient A), cardiomyopathy (patient B), LIMD (NDUFS6 deletion, patient C [15]), Leigh disease/cytochrome c oxidase (COX) deficiency (patient D), COX deficiency (SURF1 mutation, patient E), or mitochondrial

Mitochondrial translation product labeling and complex formation

Newly synthesized mtDNA-encoded subunits can be specifically labeled by incubating cells with [³⁵S]methionine/cysteine in the presence of CHX, which blocks cytosolic protein synthesis [21]. To stabilize these subunits, nuclear-encoded subunits were first accumulated in mitochondria by pretreating cells with CAP [24]. Assembly of mtDNA-encoded subunits into respiratory complexes was monitored by the further incubation of cells following the removal of radiolabel and translation inhibitors (Fig. 1

Discussion

In humans, the respiratory complexes of the mitochondrial inner membrane contain approximately 90 different subunits, with 13 encoded by mtDNA. Assembly of these complexes requires subunit expression from both mitochondrial and nuclear genomes as well as the involvement of various assembly factors, chaperones, and protein translocation components required for targeting and folding of subunits at the inner membrane. The complicated nature of this assembly has limited the generation of detailed

Acknowledgments

We thank N. Hoogenraad for discussions, J. Hoogenraad for cell culture, I. Trounce for cell lines, and A. Lombes for the ND1 antibody. This work was supported by grants from the National Health and Medical Research Council (NHMRC, 280615 and 237137) and the Muscular Dystrophy Association. M.M. is supported by an NHMRC Peter Doherty Fellowship (380840), D.R.T. is supported by an NHMRC Senior Research Fellowship (216721), and M.L. is supported by an Australian Postgraduate Award.

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Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis

Abstract

Section snippets

Cell lines and culture conditions

Mitochondrial translation product labeling and complex formation

Discussion

Acknowledgments

Biochim. Biophys. Acta

Anal. Biochem.

J. Biol. Chem.

Mol. Cell

J. Biol. Chem.

J. Mol. Biol.

Methods Enzymol.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Mol. Biol.

Biochim. Biophys. Acta

Mitochondrial diseases in man and mouse

Science

Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory-chain NADH dehydrogenase

Nature

URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit

Science