The mitochondria are ubiquitous intracellular organelles in eukaryotes, playing primary roles in metabolic processes in both healthy and diseased states, also playing a vital role in biotic and abiotic stress tolerance in plants. The mitochondria fulfil their many roles via the functionality of both nuclear- and mitochondrially- encoded proteins. This thesis is comprised of two parts, both investigating such mitochondrially-localised proteins. Polymerase-δ interacting protein 2 (POLDIP2) plays a central role in DNA replication and DNA damage repair, in-part by modulating the binding and processivity of PrimPol, an archaeo-eukaryotic primase/polymerase involved in mitochondrial and nuclear genome stability, to facilitate translesion bypass synthesis. As this interaction had previously been reported in vitro, an approach was taken to attempt to isolate a POLDIP2-PrimPol complex for in-complex structural characterisation. Full-length and truncated proteins were expressed and purified to investigate their interaction via chromatographic co-purification and chemical cross-linking. Unfortunately, these methods proved inconclusive in corroborating the previously reported interaction, and thus a stable complex could not be isolated for further characterisation. POLDIP2 is also implicated in an array of regulatory roles, including maintaining the cellular redox environment via stimulation of intracellular reactive oxygen species. A previous crystal structure highlighted a traversing hydrophilic channel, which the authors suggested may contain a potential redox-mediated cysteine 266 thiol switch. To investigate this hypothesis, the POLDIP2 cysteine mutants were purified and subjected to crystallisation and structure determination. The crystal structures of the cysteine to serine and alanine mutants were solved to 2.8 Å and 3.3 Å respectively. No significant changes in secondary or tertiary structure were observed, and there was no evidence of bound ligands within the channel. However, they did present in a different space group. Voltage dependent anion channels (VDACs) are the most abundant proteins in the mitochondrial outer membrane (OMM) and exhibit regulatory functions including in the maintenance of the cellular redox state. VDACs are a group of well-characterized pore-forming membrane proteins in eukaryotes, exhibiting established cross-species roles as multi-substrate transporters, while directing cell fate and modulating fertility, development and (in plants) physiology. Ubiquitination of VDACs has also been reported with implications in controlling cell fate in Parkinson’s disease, cancers and liver fibrosis, as well as VDAC’s ability to oligomerise. Following precedents set from the previous report of the direct interaction of Arabidopsis thaliana (At)VDAC3 with mitochondrial kinesin-like protein 1 (AtmKLP1), two native and one chimeric AtVDAC constructs, and C-terminal AtmKLP2 were cloned, expressed and purified for probing potential interactions. Due to the presence of a RING domain in AtmKLP2 and given VDAC’s reported propensity for The mitochondria are ubiquitous intracellular organelles in eukaryotes, playing primary roles in metabolic processes in both healthy and diseased states, also playing a vital role in biotic and abiotic stress tolerance in plants. The mitochondria fulfil their many roles via the functionality of both nuclear- and mitochondrially- encoded proteins. This thesis is comprised of two parts, both investigating such mitochondrially-localised proteins. Polymerase-δ interacting protein 2 (POLDIP2) plays a central role in DNA replication and DNA damage repair, in-part by modulating the binding and processivity of PrimPol, an archaeo-eukaryotic primase/polymerase involved in mitochondrial and nuclear genome stability, to facilitate translesion bypass synthesis. As this interaction had previously been reported in vitro, an approach was taken to attempt to isolate a POLDIP2-PrimPol complex for in-complex structural characterisation. Full-length and truncated proteins were expressed and purified to investigate their interaction via chromatographic co-purification and chemical cross-linking. Unfortunately, these methods proved inconclusive in corroborating the previously reported interaction, and thus a stable complex could not be isolated for further characterisation. POLDIP2 is also implicated in an array of regulatory roles, including maintaining the cellular redox environment via stimulation of intracellular reactive oxygen species. A previous crystal structure highlighted a traversing hydrophilic channel, which the authors suggested may contain a potential redox-mediated cysteine 266 thiol switch. To investigate this hypothesis, the POLDIP2 cysteine mutants were purified and subjected to crystallisation and structure determination. The crystal structures of the cysteine to serine and alanine mutants were solved to 2.8 Å and 3.3 Å respectively. No significant changes in secondary or tertiary structure were observed, and there was no evidence of bound ligands within the channel. However, they did present in a different space group. Voltage dependent anion channels (VDACs) are the most abundant proteins in the mitochondrial outer membrane (OMM) and exhibit regulatory functions including in the maintenance of the cellular redox state. VDACs are a group of well-characterized pore-forming membrane proteins in eukaryotes, exhibiting established cross-species roles as multi-substrate transporters, while directing cell fate and modulating fertility, development and (in plants) physiology. Ubiquitination of VDACs has also been reported with implications in controlling cell fate in Parkinson’s disease, cancers and liver fibrosis, as well as VDAC’s ability to oligomerise. Following precedents set from the previous report of the direct interaction of Arabidopsis thaliana (At)VDAC3 with mitochondrial kinesin-like protein 1 (AtmKLP1), two native and one chimeric AtVDAC constructs, and C-terminal AtmKLP2 were cloned, expressed and purified for probing potential interactions. Due to the presence of a RING domain in AtmKLP2 and given VDAC’s reported propensity for ubiquitination, the ability of AtmKLP2 to ubiquitinate VDAC was also investigated. This thesis provides supporting evidence for the direct interaction of AtVDACs 1 and 2 with AtmKLP2. E3-autoubiquitylation assays were also employed to explore the potential for AtmKLP2 to ubiquitinate AtVDAC2, with results suggesting the ability of AtmKLP2 to receive ubiquitin and to potentially autoubiquitinate, but inconclusive evidence as to its ability to ubiquitinate AtVDAC2. These investigations constitute a foundation for future characterisation of VDAC-KLP interactions, with the potential to investigate the molecular and electrophysiological impact of such interaction events, with possible implications in controlling VDAC oligomerisation, cell fate and fertility, as well as providing cross-species insight into the effects of VDAC-kinesin interactions.
| Date of Award | 31 Mar 2025 |
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| Original language | English |
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| Supervisor | Richard Bingham (Main Supervisor) & Natasha Reed (Co-Supervisor) |
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