Disease-causing mutations in subunits of OXPHOS complex I affect certain physical interactions.

Area of Science:

• Mitochondrial biology
• Molecular genetics
• Biochemistry

Background:

• Mitochondrial complex I (CI) is a large, multi-subunit protein complex crucial for oxidative phosphorylation (OXPHOS).
• High-resolution structures of human and mammalian CI enable prediction of mutation impacts on subunit interactions.
• Experimental validation of predicted interactions requires efficient, high-throughput methods.

Purpose of the Study:

• To create a high-throughput platform for assessing pairwise protein-protein interactions within mitochondrial complex I.
• To experimentally validate the impact of known pathogenic mutations on CI subunit interactions.
• To investigate the functional consequences of mutations at subunit interfaces.

Main Methods:

• Cloned all 37 nuclear DNA (nDNA) and 7 mitochondrial DNA (mtDNA)-encoded human CI subunits into yeast expression vectors.
• Utilized the split murine dihydrofolate reductase (mDHFR) protein complementation assay (PCA) for interaction studies.
• Employed the platform to examine reported pathological OXPHOS CI mutations at subunit interaction interfaces.

Main Results:

• Successfully established a split mDHFR PCA platform for studying CI subunit interactions in yeast.
• Demonstrated the assay's capacity to detect alterations in protein interactions.
• Identified that a pathogenic frame-shift mutation in the MT-ND2 gene leads to loss of specificity in ND2-based interactions.

Conclusions:

• The split mDHFR PCA is a powerful tool for assessing disease-causing mutations' impact on pairwise protein-protein interactions within large complexes like CI.
• This assay provides a potential mechanistic explanation for the pathogenicity of mutations affecting CI subunit interactions.
• Facilitates understanding of mitochondrial diseases linked to OXPHOS dysfunction.