Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

Understanding the roles of subunits and assembly factors implicated in the biogenesis of human Complex II (#128)

Joanna Sacharz 1 , Boris Reljic 1 , Daniella H Hock 1 , David de Souza 2 , Michael T Ryan 3 , Dedreia Tull 2 , David A Stroud 1
  1. Department of Biochemistry and Molecular Biology, Bio21, The University of Melbourne, Melbourne, VIC, Australia
  2. Metabolomics Australia, Bio21, The University of Melbourne, Melbourne, VIC, Australia
  3. Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia

We sought to explore the roles of assembly factors (SDHAF1-4) in the biogenesis and function of Complex II (CII), namely SDH (Succinate Dehydrogenase) in human cell lines. Most of our knowledge about the function of CII assembly factors comes from studies performed in the yeast S. cerevisiae. We employed gene editing using CRISPR/Cas9 to generate knockout (KO) cell lines of the four core subunits of CII (SDHA-D) and its 4 known assembly factors. We carried out a comprehensive biochemical, metabolic and proteomic analysis of each KO cell line. In order to confirm the direct interactions, we performed affinity enrichment mass-spectrometry (AE-MS) of tagged subunits.

Our data suggests that the CII core subunits SDHA, SDHC, SDHD, but not SDHB, are essential for cell viability. We show altered metabolism in a mutant of SDHA expressing <10% of the native protein, as well as strong turnover of other CII subunits. Loss of SDHB results in turnover of SDHC and SDHD, but not SDHA. Knockout of SDHAF1-4 assembly factors had no impact on the levels of CII core subunits and only minimal impact on CII assembly. Conversely loss of SDHAF1 and SDHAF3, as well as SDHA resulted in turnover of ALDH1L1 and ALDH1L2 (cytosolic and mitochondrial isoforms of 10-formyltetrahydrofolate dehydrogenase) implicated in one-carbon metabolism, and PCK2 (phosphoenolpyruvate carboxykinase) involved in the conversion of oxaloacetate (OAA) to phosphoenolpyruvate in the TCA cycle. This data suggests that CII subunits and assembly factors have yet to be elucidated roles in mammalian mitochondria.