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Analysis of mitochondrial outer membrane import complex components in Neurospora crassa.

  • Author / Creator
    Lackey, Sebastian W K
  • The TOB (Topogenesis of Outer membrane β-barrel proteins) complex is composed of the core proteins Tob55 (Sam50), Tob37 (Sam37) and Tob38 (Sam35). The complex facilitates the insertion of all β-barrel and some α-helically anchored integral proteins into the mitochondrial outer membrane (MOM). Little is understood about the architecture of the TOB complex and the protein-protein interactions required for complex stability and function. We have shown that the three TOBcore proteins are essential in Neurospora crassa making in vivo analysis of simple TOB knockout strains impossible. However, I have shown that severe depletion of Tob37 or Tob38 protein in heterokaryotic cultures leads to disruption of β-barrel protein import as well as the import of at least one α-helically anchored MOM protein, Tom22. In addition, I identified a key topological feature located near the C-terminus of Tob37. This region contains novel dual α-helices, one of which acts as the transmembrane domain (TMD) anchor for Tob37 that stabilizes the association of the protein with the complex. I have also shown that Tob38 is severely reduced in the absence of Tob37 and that Tob38 likely interacts with Tob37. In a second project I examined the topology of N. crassa Tom40, the essential core β-barrel protein of the TOM (Translocase of the Outer Membrane) complex. Tom40 forms the pore through which the vast majority of mitochondrial proteins must pass through to enter the organelle. I used substituted cysteine accessibility mapping (SCAM) to define the topology and identify the structural β-strand arrangement of specific amino acid residues in a region of Tom40. These data, together with previous data from our laboratory allowed the development of a partial model based on empirical molecular evidence that we compared to various in silico developed models. The analysis favors the recently proposed model of a β-barrel protein containing 19 β-strands that was developed in other laboratories using the structure of the related protein VDAC (or porin) as a model.

  • Subjects / Keywords
  • Graduation date
    2014-06
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3595D
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biological Sciences
  • Specialization
    • Molecular Biology and Genetics
  • Supervisor / co-supervisor and their department(s)
    • Nargang, Frank (Biological Sciences)
  • Examining committee members and their departments
    • Stuart, Rosemary (Biological Sciences, Marquette University)
    • Raivio, Tracy (Biological Sciences)
    • Locke, John (Biological Sciences)
    • Lemire, Bernard (Biochemistry)