Functional and mechanistic studies of human mitochondrial carriers

The SLC25 mitochondrial carrier family is the largest family of transporters, with 53 members in humans. They selectively transport a diverse set of substrates across the mitochondrial inner membrane, and are involved in key aspects of mitochondrial metabolism and function. The identification and characterisation of human mitochondrial carriers is essential for the understanding of these proteins in the context of human health and disease. There are many unanswered questions and contentious issues with regards to the members of the SLC25 family, including the verification of proposed substrates, the characterisation of transport activity, substrate binding and proton coupling, and the kinetic mechanism. The aim of this thesis was to address some of these controversies by understanding the properties of specific proteins, and by extension the mitochondrial carrier family as a whole. To that end, the verification of SLC25A51 as a mitochondrial NAD carrier was attempted. However, transport assays in L. lactis were not successful, and expression of the protein in S. cerevisiae and purification was challenging. The human mitochondrial phosphate carrier was expressed and purified for the first time, and aspects of substrate specificity were addressed. A library of twelve alanine mutants was made with the aim of determining the substrate binding site by thermal shift analysis. The results obtained provided a proof of principle. Finally, the kinetic mechanism of mitochondrial carriers, which was claimed to be sequential, was revisited. This was the only extant evidence in favour of a dimer model. Two-reactant initial-velocity experiments were performed with the human ADP/ATP, oxoglutarate, dicarboxylate, aspartate/glutamate, and Mg-ATP/Pi carriers using robotic transport assays. It was found that, contrary to the literature, mitochondrial carriers operate with a ping-pong kinetic mechanism in which one substrate is transported and released from the protein before the counter-substrate binds for transport in the opposite direction. This kinetic mechanism is consistent with mitochondrial carriers being monomers with a single substrate binding site operating with an alternating access mechanism.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Cimadamore-Werthein, Camila

Advisor dc:contributor.advisor

• Kunji, edmund

• Kinetic mechanism
• Mitochondrial carriers

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/06257e4b-f936-4b5a-acbf-0ccf11ff09ea/download
• https://www.rioxx.net/licenses/all-rights-reserved/

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.106435

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/364978