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Functional characterisation of the human mitochondrial ATP-Mg/Pi carriers and their disease-causing variants



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Fitzpatrick, Fiona Mary 


The mitochondrial ATP-Mg/Pi carriers (APCs) catalyses the exchange of adenine nucleotides for phosphate across the mitochondrial inner membrane, which is regulated by calcium. They are responsible for modulating the adenine nucleotide pool in the mitochondrial matrix, and therefore have a pivotal role in meeting the energetic demands of the cell. They consist of three domains; an EF hand-containing regulatory domain responsive to extra-mitochondrial calcium, an amphipathic helix, and a mitochondrial carrier domain, which catalyses substrate transport. In the proposed “locking-pin” mechanism, the amphipathic helix is bound to the regulatory domain in the presence of calcium, whereas it is bound to the carrier domain in the absence of calcium. There are four different human APC paralogues: APC1, APC2, APC3 and APC4, some of which have multiple splice variants. Recently, disease variants in APC1 and APC3b have been identified in patients with severe developmental disease or with kidney dysfunction, respectively. The biochemical properties of human paralogues APC2, APC3 and APC4 have not been investigated, and elucidation of their transport kinetics and the regulatory mechanism may provide useful insights into their role in cellular physiology. The aim of the thesis was to characterise these paralogues at the molecular level with regards to substrate transport and calcium regulation. Heterologous expression and purification of the proteins in a folded and stable form allowed their basic properties to be defined. Thermal stability assays were used to assess the quality of the protein and to probe the calcium binding mechanism, indicating that APC2 and APC3b also have a “locking-pin” mechanism, as exhibited by APC1. Activity assays with proteoliposomes showed that APC2 and APC3b also transported adenine nucleotides and were regulated by calcium. However, the transport activities and response to calcium differed between the paralogues. APC2 has a high transport rate and an on/off switch for calcium regulation, whereas APC1 has a lower transport rate and a dimmer switch for calcium regulation, and APC3b is between. A combined approach using APC chimeric proteins and regulatory domain truncations was used to identify which functional domain(s) were responsible for the observed differences. The results pointed towards the amphipathic helix, and its interaction with the regulatory or carrier domain, although further investigations are required. Another set of aims was to assess the effect of pathogenic variants on the activity of APCs to demonstrate their link to disease. The R217C and R217H mutations in APC1 are linked to Gorlin-Chaudhry-Moss and Fontaine Progeroid syndrome, which severely affect development. A Q349H mutation in APC3b was linked to the development of kidney stones through whole exome sequencing analysis. The analyses showed that these mutations most likely disrupt a key interaction in the domain structure, affecting the overall stability of the carrier and reduce the overall transport rates by about half. The assays established herein allow the properties of the APCs to be investigated further and provide a means to assess the effect of identified pathogenic variants on the function of the proteins.





Kunji, Edmund RS


Mitochondria, Mitochondrial transport proteins, Calcium regulation, Disease variants, Rare diseases


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
MRC (1659316)
Medical Research Council (MC_UU_00015/1)
MRC (1659316)