Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Investigation of the Mitochondrial Translocase of the Outer Membrane (TOM) of Drosophila melanogaster (#203)

Agalya Periasamy 1 , Naomi Mitchell 2 , Arjun S Chahal 2 , Thomas Bausewein 3 , Werner Kühlbrandt 3 , Leonie M Quinn 2 , Jacqueline M Gulbis 1 4
  1. Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria, Australia
  2. Cancer Biology and Therapeutics, The John Curtin School of Medical Research, Canberra, ACT, Australia
  3. Structural Biology Division, Max Planck Institute of Biophysics, Frankfurt, Germany
  4. Department of Medical Biology, The University of Melbourne, Parkville, Melbourne, Victoria, Australia

A vast majority of mitochondrial proteins are nuclear-encoded and are imported into the organelle from the cytosol as newly synthesized precursors. The Translocase of the Outer Membrane (TOM) is a multi-subunit protein assembly that serves as the general entry gate for all incoming precursors. So far, structural investigations on TOM have been restricted to the ascomycete fungi (Saccharomyces and Neurospora) from which the complex(es) can be purified from native membranes in sufficient quantities for single particle electron microscopy (EM) studies.The mitochondrial proteome of Drosophila is evolutionarily closer to humans and its TOM components exhibit high sequence homology to their human counterparts. In order to investigate the structure and membrane organization of the Drosophila TOM complex, we utilised the fruit-fly, Drosophila melanogaster for expression and analysis of the biology in vivo. Targeting the expression of tagged Tom40 to Drosophila eyes enabled purification of DmTOM for single particle EM. Unlike recent structures of TOM1, 2, the Drosophila complex contains Tom20, the presequence receptor. Preliminary analysis by single particle negative stain and cryo-EM reveals three-pore particles expected for the holo-TOM complex. Tryptic-digest mass spectrometric analysis of purified TOM has also enabled us to identify the Drosophila orthologues of Tom5 and Tom6 and other mitochondrial proteins that associate with TOM.