Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Functional characterisation of HIGD1A and HIGD2A and their roles in the assembly of mitochondrial respiratory chain (#261)

Daniella H Hock 1 2 , Joanna Sacharz 1 2 , Boris Reljic 1 2 , David A Stroud 1 2
  1. University of Melbourne, Melbourne, VIC, Australia
  2. Department of Biochemistry and Molecular Biology, University of Melbourne, Bio21 Molecular Science and Biotechnology Institute, Parkville, VIC, Australia

Mitochondria generate over 95% of cellular ATP through a series of reactions across five inner mitochondrial membrane complexes (Complex I-V) known as the respiratory chain. Together, these multi-protein complexes are comprised of over 100 proteins, which are assembled with the aid of proteins called assembly factors. In addition to being found in holocomplexes, Complexes I, III and IV can also assemble into higher order structures called supercomplexes. Mutations in many genes encoding assembly factors cause mitochondrial disease through misassembled complexes and therefore impaired ATP production. HIGD1A and HIGD2A are mammalian homologs of Rcf1, which in yeast is thought to function as supercomplex assembly factor and regulator of Complex IV activity. We sought to investigate the function of mammalian HIGD1A and HIGD2A by generating knockout cell lines in human HEK293T using CRISPR/Cas9. Assembly of mitochondrial respiratory chain complexes were investigated by BN-PAGE. Stable isotope labelling with amino acids in cell culture (SILAC) and quantitative proteomics was performed to measure the relative levels of cellular and mitochondrial proteins. Rescue of the knockout phenotype was performed using a FLAG-tagged construct, which also allowed interactors to be determined by affinity enrichment mass spectrometry (AE-MS). Translation and turnover of mitochondrial DNA (mtDNA)-encoded subunits was investigated using a novel pulse SILAC analysis. Hypoxia was induced by incubation of cells in 1% O2and 5% CO2for 18 hours. As results, HIGD1AKOhad no effect on the assembly of respiratory complexes in normal oxygen conditions while grown of the cells in an hypoxic environment led to decreases in the abundance of mitochondrial proteins compared to control. HIGD2AKOcaused impaired function of mitochondrial Complex IV by destabilization of the COX3 module, a step of assembly for which no assembly factors have been previously discovered. Hence, our studies provide insights into the role of HIGD1A and HIGD2A within the mitochondria and characterise HIGD2A as an assembly factor and a potential novel mitochondrial disease gene.