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Permanent link (DOI): https://doi.org/10.7939/R3595D

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

Descriptions

Other title
Subject/Keyword
protein import
Neurospora crassa
mitochondria
Tom40
TOB
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Lackey, Sebastian W K
Supervisor and department
Nargang, Frank (Biological Sciences)
Examining committee member and department
Lemire, Bernard (Biochemistry)
Stuart, Rosemary (Biological Sciences, Marquette University)
Locke, John (Biological Sciences)
Raivio, Tracy (Biological Sciences)
Department
Department of Biological Sciences
Specialization
Molecular Biology and Genetics
Date accepted
2014-01-23T08:36:51Z
Graduation date
2014-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R3595D
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Lackey, S.W., Wideman, J.G., Kennedy, E.K., Go, N.E., and Nargang, F.E. (2011). The Neurospora crassa TOB complex: analysis of the topology and function of Tob38 and Tob37. PLoS One 6, e25650.

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