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Title: An in vivo approach to elucidating the function of mitochondrial porin by the characterisation of Neurospora crassa strains deficient in porin
Authors: Summers, William A T
Supervisor: Court, Deborah (Microbiology)
Examining Committee: Vierula, John (Carelton University) Wilkins, John (Biochemistry and Medical Genetics) Loewen, Peter (Microbiology) Hausner, Georg (Microbiology)
Graduation Date: October 2010
Keywords: Neurospora
Issue Date: 10-Sep-2010
Citation: Summers, W. A. and Court, D. A. (2010). "Origami in outer membrane mimetics: correlating the first detailed images of refolded VDAC with over 20 years of biochemical data." Biochem Cell Biol 88(3): 425-38.
Abstract: The mitochondria are the primary energy providers for most eukaryotic cells. The substrate and products of the mitochondria need to be translocated across the semi-permeable mitochondrial outer membrane (MOM). Mitochondrial porin is an aqueous channel in the MOM thought to provide the primary pathway for metabolite translocation. Porin is a nuclear encoded protein and therefore needs to be transported to the mitochondria, translocated across and assembled within the MOM. Of all the recognition signals required for successful transport, import and assembly, only the β-sorting signal used in assembly is known. In addition, this protein possesses the ability to gate, and in doing so can preferentially allow the passage of anions in the open state and cations in the closed state. However, the precise mechanism by which gating of porin occurs and a complete understanding of porin’s function in vivo remains elusive. The essentiality of porin was examined by constructing a strain of Neurospora crassa deficient for porin. This strain, denoted as WS004, exists as evidence that porin is non-essential for the survival of Neurospora crassa. However, the loss of porin results in a reduction in growth rate due to the dysfunction of the cytochrome mediated respiratory pathway, which was made evident by the reduction of cytochrome b and almost complete lack of cytochrome aa3. WS004 survives by inducing the expression of alternative oxidase, which funnels the electrons from the Q pool directly to oxygen, bypassing the cytochrome b and aa3 containing complexes III and IV respectively. Additional phenotypic differences observed included loss in ability to produce aerial hyphae, reduced amount of conidia produced and strains that were female sterile. It was determined, that additional genetic factors influenced the resulting phenotype due to the loss of porin. LC-MS/MS, in combination with iTRAQ labelling, was utilized to examine changes in the proteome profiles of porin containing and porin lacking mitochondria and showed several different proteins as significantly up- or down-regulated which lend to an explanation to some of the phenotypes observed. Taken together, these results demonstrate the central role of porin in regulating both mitochondrial and cellular processes.
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