Abstract
Mitochondrial uncoupling proteins (UCPs) attenuate proton motive force across the inner membrane of mitochondria (IMM) by transporting protons from the intermembrane space into matrix, thus reducing the ATP synthesis rate. UCPs together with adenine nucleotide translocase (ANT) and inorganic phosphate translocase (PiT) create a subgroup of carriers directly linked to mitochondrial energetics. While its potential role in neuroprotection of neurogenerative diseases has been speculated, the brain specific neuronal UCP4 is among the least investigated UCPs. This integrative study will focus on the structural features and functional interrelation of UCP4, ANT and PiT. We designed recombinant versions of these proteins that, using a modified autoinduction method, were overexpressed in the membrane of Escherichia coli, extracted with mild detergents, and purified as monomers. Purified proteins, embedded in a shell of bacterial membrane lipids, were then reconstituted in IMM-mimicking phospholipid vesicles for biophysical studies using circular dichroism (CD) and fluorescence spectroscopies. Native-like helical conformations of proteins in the mild detergent octyl glycoside (OG) and phospholipid membranes were confirmed by CD spectroscopy. Interestingly, in agreement with our previous studies, monomeric UCP4 spontaneously self-associated to form tetramers in lipid vesicles. Conversely, ANT and PiT maintained their molecular forms in both detergent and liposome milieus. Reconstituted proteins in lipid membranes exhibited different levels of proton transport activity in the presence of fatty acids and decreased with the addition of ATP. UCP4 showed significantly higher proton transport rates compared to ANT and PiT. Research on the functional interrelation between these three carrier proteins, as well as the effect of mitochondrial lipid cardiolipin on their conformational stability and proton transport activity, is in progress in our laboratory.
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