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dc.contributor.authorMarino, Polly
dc.date.accessioned2019-02-14T08:55:04Z
dc.date.available2019-02-14T08:55:04Z
dc.date.issued2019-03-30
dc.date.submitted2018-09-25
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/289386
dc.description.abstractComplex I (NADH:ubiquinone oxidoreductase) couples electron transfer to proton translocation across the inner mitochondrial membrane, to drive the synthesis of ATP. Its distinctive L-shaped structure comprises 45 subunits, encoded by both the mitochondrial and nuclear genomes, which are assembled by a complicated modular pathway. Complex I genetic defects are the most common cause of mitochondrial disorders and often present in early childhood, with high mortality rates. Recent high-resolution electron cryo-microscopy structures of mammalian complex I provide a foundation for both interpreting biochemical and biomedical data and understanding the catalytic mechanism. First, this thesis explores how the flavin cofactor is inserted into the NADH-binding (N-) domain of complex I. Genetic manipulation of cultured human cells, to starve them of flavin, revealed a hierarchal impact on the mitochondrial flavoproteome. High riboflavin content in the growth media ameliorated observed phenotypes, requiring cell conditioning in low riboflavin conditions. CRISPR knockout of the putative mitochondrial flavin transporter SLC25A32 demonstrated the severe impact of decreased flavin on complexes I and II, and mass spectrometry ‘complexome’ analyses suggest that the N-domain is still assembled onto complex I in the absence of the flavin. Second, the model organism Yarrowia lipolytica was used to assess the importance of residues in the quinone-binding site of complex I. Three residues with proposed roles in binding the quinone head-group were targeted. One variant was catalytically inactive, while two retained some activity. They showed decreased ability to reduce physiologically-relevant, long chain quinones, but their ability to reduce short-chain analogues was affected less severely. The results suggest a complicated picture in which interactions between the protein and both the hydrophilic quinone head-group and hydrophobic isoprenoid chain contribute to quinone-binding affinity and catalysis. Finally, a model for human complex I, generated from a recent high-resolution structure of mouse complex I, was used to investigate whether the pathogenicity of human variants could be predicted. Structural information on variant residues, including their secondary structure, proximity to key features and surface exposure, was collated and the power of each property to predict pathogenicity investigated. The analysis was then extended to the whole structure, to identify potential pathogenic hotspots in the enzyme, inform future studies of functionally important regions in complex I, and aid the diagnosis of clinically relevant pathogenic variants.
dc.language.isoen
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectcomplex I
dc.subjectmitochondria
dc.subjectrespiration
dc.subjectyarrowia
dc.subjectflavin
dc.subjectassembly
dc.subjectriboflavin
dc.subject143B
dc.subjectSILAC
dc.subjectcomplexome
dc.subjecttranscriptomics
dc.subjectSLC25A32
dc.subjectRFK
dc.titleStudies on assembly and genetic variation in mitochondrial respiratory complex I
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.publisher.departmentMRC Mitochondrial Biology Unit
dc.date.updated2019-01-04T17:22:52Z
dc.rights.generalAll contributions by other researchers are acknowledged in the thesis
dc.identifier.doi10.17863/CAM.36634
dc.publisher.collegeCorpus Christi
dc.type.qualificationtitlePhD in Biological Science
cam.supervisorHirst, Judy
cam.thesis.fundingtrue
rioxxterms.freetoread.startdate2020-02-14


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Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
Except where otherwise noted, this item's licence is described as Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)