The role of matrix AAA+ proteases in protein homeostasis in mammalian mitochondria

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry, La Trobe Institute for Molecular Science (LIMS), School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Mitochondria are essential eukaryotic organelles responsible for key cellular processes such as energy conversion, the biosynthesis of specialised molecules and the regulation of cellular signalling pathways. The many roles of mitochondria depend on a functional population of enzymes and other proteins that exist at a high concentration in this distinct cellular entity. Mitochondrial protein homeostasis, is largely maintained by molecular chaperones and proteases. While molecular chaperones assist protein folding, the proteases degrade non-functional and unwanted proteins maintaining protein homeostasis and preventing potentially toxic protein aggregation. However the precise role of the proteases LONM and CLPXP in the mitochondrial matrix of mammals is poorly defined. This study revealed that turnover of an aggregation prone mitochondrial protein was performed primarily by the matrix protease, LONM. Moreover, using in vitro degradation assays with purified recombinant components, LONM was shown to be sensitive to the conformational state of a substrate, as it was unable to degrade native or aggregated proteins but could rapidly degrade several unfolded proteins. To obtain further insight into the role of matrix proteases in mitochondria, the turnover of a mutant form of mitochondrial SDH5, which causes hereditary paraganglioma 2, was also investigated. Wild type SDH5 was stable in mitochondria, while the SDH5G78R mutant was rapidly degraded by LONM. In contrast, both wild type and mutant SDH5, in the absence of the interaction partner SDHA, were rapidly degraded by LONM in vitro. These data suggest that surface exposed residues on SDH5, normally buried in the SDH5- SDHA interface are required for LONM-mediated degradation. The compromised SDH5-SDHA interaction results in rapid degradation of SDH5G78R by LONM potentially exacerbating the disease state. The improved molecular understanding of the matrix proteases, as described in this study, will be helpful in the development of therapeutic interventions for mitochondrial diseases.