Understanding the assembly of mitochondrial machines


Project Details

In humans, OXPHOS takes place on the five membrane protein complexes comprising the respiratory chain. These large multi-subunit complexes are together comprised of 93 subunits that are encoded by both mitochondrial (mt) and nuclear DNA. Mutations in all mtDNA protein coding genes and >180 nuclear genes impair OXPHOS and cause classical mitochondrial disease, however many of these genes do not encode subunits of the respiratory chain but rather proteins involved in OXPHOS biogenesis known as assembly factors. Although we have a good understanding of the enzymatic roles of each complex in OXPHOS, we still do not fully understand how the system is built and maintained, nor the specific roles of many subunits and assembly factors in this process.

With our collaborator Professor Mike Ryan (Monash University) we developed a novel systems approach, coupling CRISPR/Cas9 based gene-editing with quantitative proteomics tools, to study the assembly pathway of complex I and discover new assembly factors (Stroud et al., Nature 2016). Our lab is now applying this approach to the remaining members of the respiratory chain, complexes II-V (Figure 1).

scheme showing concominant assembly for each KO.
Figure 1: (A) Analysis of CRISPR/Cas9 generated KO cell lines for a subunit of each respiratory complex I-V by Blue-Native (BN)-PAGE & immunoblotting. Concominant assembly defects in complex assembly can be seen for each KO. (B) Subunit levels for KO’s in B determined by quantitative mass-spectrometry and mapped onto the structures of complexes I-V. Yellow and arrow correspond to the KO subunit and reduced stability of subunits correlate with the blue intensity. PDBs: CI, 5LDW; CII, 1ZOY; CIII, 1BGY; CIV, 5B1A; CV, 5ARE.

In addition to extensive gene-editing and quantitative proteomics tools, this project relies heavily on classical molecular techniques such as mammalian cell culture, Blue-Native (BN) PAGE, western blotting, affinity enrichment techniques such as co-immunoprecipitation and BioID, cellular imaging, computational and structural biology, and metabolic measurements such as oxygen consumption and ATP production.

Research Group

Stroud laboratory: Mitochondrial Systems Biology

Faculty Research Themes

Child Health

School Research Themes

Cardio-Respiratory, Systems Biology, Molecular Mechanisms of Disease

Key Contact

For further information about this research, please contact the research group leader.

Department / Centre

Biochemistry and Molecular Biology

Unit / Centre

Stroud laboratory: Mitochondrial Systems Biology

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