Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Abstract

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F 0 F 1 -ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F 0 F 1 -ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F 0 F 1 -ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production. mitochondrial membrane proteins | proton diffusion | direct stochastic optical reconstruction microscopy | uncoupling | reactive oxygen species M itochondria are involved in a wide range of cell functions, including fatty acid oxidation, calcium homeostasis, apoptosis, reactive oxygen species (ROS) signaling, and above all, production of ATP (1, 2). In neurons, these organelles are transported along neuronal processes to provide energy for areas of high energy demand, such as synapses (3). To support their functions, mitochondria exhibit a complex morphology consisting of separate and functionally distinct outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). The latter is structurally organized into two domains: an inner boundary membrane (IBM) and a cristae membrane (CM) (4). The current hypotheses imply that the morphology/topology of the IMM is tightly related to biochemical function, the energy state, and the pathophysiological state of mitochondria (5). Whereas the OMM contains porins [e.g., voltage-dependent anion channel (VDAC)], which mediate its permeability to molecules up to 10 kDa, the IMM topology is highly complex. It is comprised of different transport proteins, the ATP synthase (complex V), and complexes I, III, and IV of the electron transport chain, which are responsible for generating the proton motive force (pmf); pmf represents the driving force for not only ATP synthesis, but also other protein-mediated transport activities (for example, phosphate, pyruvate, and glutamate transport). Uncoupling protein 1 (UCP1; thermogenin), a memb...

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Origin of proton affinity to membrane/water interfaces

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