Background:The phospholipid cardiolipin undergoes acyl chain remodeling after biosynthesis, which has been hypothesized to optimize mitochondrial function. Results: ⌬cld1 yeast, containing unremodeled cardiolipin, have no mitochondrial morphology or oxidative phosphorylation defects. Conclusion: Cardiolipin remodeling is not required for optimal mitochondrial bioenergetic function in yeast. Significance: Cardiolipin remodeling may be important for presently unknown mitochondrial processes and/or have unappreciated physiological functions.

The anionic phospholipid cardiolipin has an unusual dimeric structure with a two-phosphate headgroup and four acyl chains. Cardiolipin is present in energy-transducing membranes that maintain electrochemical gradients, including most bacterial plasma membranes and the mitochondrial inner membrane, where it mediates respiratory complex assembly and activation, among many other roles. Dysfunctional biogenesis of cardiolipin is implicated in the pathogenesis of several diseases including Barth syndrome. Because cardiolipin is a dominant anionic lipid in energy-conserving membranes, its headgroup is a major contributor to surface charge density and the bilayer electrostatic profile. However, the proton dissociation behavior of its headgroup remains controversial. In one model, the pKa values of the phosphates differ by several units and the headgroup exists as a monoanion at physiological pH. In another model, both phosphates ionize as strong acids with low pKa values and the headgroup exists in dianionic form at physiological pH. Using independent electrokinetic and spectroscopic approaches, coupled with analysis using Gouy–Chapman–Stern formalism, we have analyzed the ionization properties of cardiolipin within biologically relevant lipid bilayer model systems. We show that both phosphates of the cardiolipin headgroup show strong ionization behavior with low pKa values. Moreover, cardiolipin variants lacking structural features proposed to be required to maintain disparate pKa values – namely the secondary hydroxyl on the central glycerol or a full complement of four acyl chains – were shown to have ionization behavior identical to intact cardiolipin. Hence, these results indicate that within the physiological pH range, the cardiolipin headgroup is fully ionized as a dianion. We discuss the implications of these results with respect to the role of cardiolipin in defining membrane surface potential, activating respiratory complexes, and modulating membrane curvature.

Mitochondrial dysfunction underlies many heritable diseases, acquired pathologies, and aging-related declines in health. Szeto–Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that are efficacious toward a wide array of mitochondrial disorders and are believed to target mitochondrial membranes because they are enriched in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, using biophysical and computational approaches, we have analyzed the interactions of the lead compound SS-31 (elamipretide) with model and mitochondrial membranes. Our results show that this polybasic peptide partitions into the membrane interfacial region with an affinity and a lipid binding density that are directly related to surface charge. We found that SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it did cause saturable alterations in lipid packing. Most notably, SS-31 modulated the surface electrostatics of both model and mitochondrial membranes. We propose nonexclusive mechanisms by which the tuning of surface charge could underpin the mitoprotective properties of SS-31, including alteration of the distribution of ions and basic proteins at the interface, and/or modulation of bilayer physical properties. As a proof of concept, we show that SS-31 alters divalent cation (calcium) distribution within the interfacial region and reduces the energetic burden of calcium stress in mitochondria. The mechanistic details of SS-31 revealed in this study will help inform the development of future compound variants with enhanced efficacy and bioavailability.

Tim23, the central subunit of the TIM23 protein-translocation complex, forms a voltage-gated channel in the mitochondrial inner membrane (MIM), an energy-conserving membrane that generates a proton-motive force to drive vital processes. Using high-resolution fluorescence mapping of a channel-facing transmembrane segment (TMS2) of Tim23 from Saccharomyces cerevisiae, we demonstrate that changes in the energized state of the MIM cause marked structural alterations in the channel region. In an energized membrane, TMS2 forms a continuous α-helix that is inaccessible to the aqueous intermembrane space (IMS). Upon depolarization, the helical periodicity of TMS2 is disrupted, and the channel becomes exposed to the IMS. Kinetic measurements confirm that changes in TMS2 conformation coincide with depolarization. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins coupled to processes such as channel gating.

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