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Investigation of the final steps in mitochondrial complex I biogenesis

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posted on 2023-01-18, 16:24 authored by Xiaonan Wang
Submission note: A thesis submitted in the total fulfilment of the requirement for the degree of Doctor of Philosophy to the Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria.

Mitochondrial complex I is the first enzyme complex of the oxidative phosphorylation system. In order to achieve its enzymatic function in human mitochondria, a total number of 44 subunits need to be assembled correctly to form the mature 980 kDa complex. Studies into complex I biogenesis have outlined this process by identifying a number of assembly intermediates and several assembly factors that are involved in this process. However to fully understand complex I biogenesis, a detailed characterisation of each assembly intermediate and a function study of each assembly factor is still required. The work presented in this thesis focuses on the characterisation of proteins involved in the late stage of complex I biogenesis. Fibroblasts from Leigh Syndrome patients with mutations in NDUFAF2 leading to complex I deficiency were utilized to characterise Ndufaf2. Analysing protein assembly profiles over time, an ~830 kDa subcomplex was noticed undergoing rapid biogenesis. This complex was then found to be unstable as it disassembled into a ~600 kDa complex containing the majority of membrane located complex I subunits. The results demonstrate that Ndufaf2 plays a key role in stabilising the ~830 kDa assembly intermediate and aids its progression into mature complex I. Further analysis revealed a putative ATP/GTP binding motif in Ndufaf2 sequence, however, it appears dispensable for its function in complex I biogenesis. Interestingly, the generation and characterisation of cells lacking the complex I subunit NDUFA9 also revealed a similar defective complex I assembly phenotype with Ndufaf2 patient fibroblasts. Analyses revealed that NDUFA9 is a structural subunit that stabilises the junction between the membrane and matrix arm of complex I. Additionally, results also indicate that the incorporation NDUFA9 follows the association of Ndufaf2 with the ~830 kDa intermediate. The biogenesis of complex is a very delicate process; the malfunction of any subunits or assembly factors can lead to its mis-assembly. This thesis demonstrates that defective cell line studies are an effective method for investigating protein interaction and function, and their impact upon complex I biogenesis.

History

Center or Department

La Trobe Institute for Molecular Science. Department of Biochemistry.

Thesis type

  • Ph. D.

Awarding institution

La Trobe University

Year Awarded

2014

Rights Statement

The thesis author retains all proprietary rights (such as copyright and patent rights) over the content of this thesis, and has granted La Trobe University permission to reproduce and communicate this version of the thesis. The author has declared that any third party copyright material contained within the thesis made available here is reproduced and communicated with permission. If you believe that any material has been made available without permission of the copyright owner please contact us with the details.

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