EXPLORING THE INTERCONNECTIONS BETWEEN MITOCHONDRIAL CARRIER PROTEINS INVOLVED IN ENERGY TRANSFER: THE CASE OF UNCOUPLIG PROTEINS 2 AND 4, ADENINE NUCLEOTIDE TRANSLOCASE, AND PHOSPHATE TRANSLOCASE

Mitochondrial carrier (MC) proteins play an essential role in cellular energy metabolism by mediating the transport of solutes and protons across the mitochondrial inner membrane (MIM). This thesis explores the structural and functional characteristics of three groups of MC proteins—Uncoupling Proteins (UCPs), Adenine Nucleotide Translocase (ANT), and Phosphate Translocase (PiT)—to elucidate their individual and interrelated cooperative roles in mitochondrial proton transport and energy regulation. The study develops and applies novel methodologies for the overexpression, purification, and reconstitution of MC proteins into model lipid bilayers mimicking the native mitochondrial inner membrane environment. These methods ensure proteins maintain their native-like conformations and functionality, enabling detailed structural and functional biophysical analyses. Results demonstrate that once reconstituted in lipid bilayers, UCP2 and UCP4 predominantly exist as tetramers, with cardiolipin (CL) significantly enhancing their proton transport activity. Conversely, ANT and PiT were observed primarily in monomeric or dimeric states, with CL stabilizing their structural forms and enhancing their proton transport efficiency. Functional assays revealed important insights into the cooperative dynamics of these proteins. Binary and ternary combinations of UCPs, ANT, and PiT formed heterotetramers. Heterotetramers from binary combinations influenced proton transport activity in distinct ways. Notably, those formed by UCP4 and ANT1, in varying stoichiometric ratios, exhibited synergistic effects on proton transport. Further investigations using inhibitors such as ATP, GTP, and Carboxyatractyloside (CATR) elucidated the complex interplay between these proteins, highlighting the impact of the lipid composition of the membrane environment, fatty acid activators, and stoichiometric ratios of the proteins on their functionality. The findings of this research address significant gaps in understanding how MC proteins may interact within the MIM to regulate energy transfer, oxidative phosphorylation, and proton leak. By characterizing their structural-functional relationships and synergistic behaviors, this work advances the broader field of mitochondrial bioenergetics and offers insights into potential therapeutic strategies for metabolic and mitochondrial-related dysfunctions.