Mitochondrial Phosphate Carrier Research Articles

The mitochondrial phosphate carrier TbMCP11 is essential for mitochondrial function in the procyclic form of Trypanosoma brucei

Conserved amongst all eukaryotes is a family of mitochondrial carrier proteins (SLC25A) responsible for the import of various solutes across the inner mitochondrial membrane. We previously reported that the human parasite Trypanosoma brucei possesses 26 SLC25A proteins (TbMCPs) amongst which two, TbMCP11 and TbMCP8, were predicted to function as phosphate importers. The transport of inorganic phosphate into the mitochondrion is a prerequisite to drive ATP synthesis by substrate level and oxidative phosphorylation and thus crucial for cell viability. In this paper we describe the functional characterization of TbMCP11. In procyclic form T. brucei, the RNAi of TbMCP11 blocked ATP synthesis on mitochondrial substrates, caused a drop of the mitochondrial oxygen consumption and drastically reduced cell viability. The functional complementation in yeast and mitochondrial swelling experiments suggested a role for TbMCP11 as inorganic phosphate carrier. Interestingly, procyclic form T. brucei cells in which TbMCP11 was depleted displayed an inability to either replicate or divide the kinetoplast DNA, which resulted in a severe cytokinesis defect.

NaStEP, an essential protein for self-incompatibility in Nicotiana, performs a dual activity as a proteinase inhibitor and as a voltage-dependent channel blocker

The S-specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner, but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S-specific pollen rejection in Nicotiana.

Loss of mitochondrial phosphate carrier SLC25A3 in skeletal muscle provides evidence for compensatory but lower capacity phosphate uptake into mitochondria

Loss of mitochondrial phosphate carrier SLC25A3 in skeletal muscle provides evidence for compensatory but lower capacity phosphate uptake into mitochondria

Loss of mitochondrial phosphate carrier in skeletal muscle: dissociation of muscle dysfunction from lower ADP phosphorylating potential

Many mutations in the mitochondrial genome and in nuclear genes that encode mitochondrial proteins hamper oxidative phosphorylation (oxphos) and lead to tissue dysfunction. Yet, whether an energy deficit is necessarily a direct cause of tissue dysfunction is not clear. We addressed this question in mice in which the mitochondrial phosphate carrier, PiC, was depleted from skeletal muscle (skm) by tamoxifen‐induced excision of nuclear gene SLC25A3 encoding PiC (Cre+). Three weeks after tamoxifen injections, PiC protein in skm was <5% of levels found in control (Ctrl) wild‐type mice (tamoxifen‐treated). Oxphos, measured in mitochondria isolated from skm, was greatly suppressed to <2%, 35% or 50% of Ctrl, depending on substrate, with essentially no oxphos when succinate (+rotenone) was supplied and the highest residual oxphos when fatty acid substrate and malate were supplied; the residual oxphos may be driven by alternate Pi uptake on the dicarboxylate carrier. Residual oxphos was similar at 3 and 9 wks of PiC depletion, suggesting that the oxphos defect was stable for this interval. Furthermore, subunits of the oxphos machinery were robustly upregulated (50–70% rise) at both time points. Nonetheless, treadmill running capacity worsened during this time. Time‐to‐exhaustion was ~50 mins in Cre+ mice with PiC absent for 3 wks, but ~25 mins when PiC was absent for 9 wks, whereas Ctrl mice ran for the scheduled 80 mins. Thus, deteriorating treadmill running in Cre+ mice seems unrelated to suppressed oxphos per se. Stress signaling via transcription factor ATF4 as well as elevated mTORC1 signaling have been associated with oxphos defects caused by mitochondrial DNA depletion in cell models and in mouse skm. These models have also suggested a causal relationship between persistent mTORC1 activation/stress signaling and cell dysfunction. Yet we found little evidence for stress signaling via ATF4 (or ATF2, ATF3, or ATF5) after 3 or 9 wks of PiC depletion, and evidence for only mild mTORC1 activation after 9 wks of PiC loss. Overall, these observations suggest that skm dysfunction is not related to an oxphos deficit in a simple way. Rather, the oxphos deficit may drive adaptive mechanisms that either become exhausted or turn maladaptive when ongoing over a longer term, or trigger additional processes that are deleterious for muscle function. Furthermore, the model presented here suggests that a mechanism other than stress signaling through ATFs is responsible for tissue dysfunction in the context of a severe oxphos defect.Support or Funding InformationFunding: R01 GM123771 to ELSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of Mitochondrial Phosphate Carrier in Skeletal Muscle: Dissociation of Muscle Dysfunction from Lower ADP Phosphorylating Potential

Loss of Mitochondrial Phosphate Carrier in Skeletal Muscle: Dissociation of Muscle Dysfunction from Lower ADP Phosphorylating Potential

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis

Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.

Inhibiting mitochondrial phosphate transport as an unexploited antifungal strategy.

The development of effective antifungal therapeutics remains a formidable challenge due to the close evolutionary relationship between humans and fungi. Mitochondrial function may present an exploitable vulnerability due to its differential utilization in fungi and its pivotal roles in fungal morphogenesis, virulence, and drug resistance already demonstrated by others. We now report mechanistic characterization of ML316, a thiohydantoin which kills drug-resistant Candida species at nanomolar concentrations through fungal-selective inhibition of the mitochondrial phosphate carrier Mir1. We established ML316 as the first Mir1 inhibitor using genetic, biochemical, and metabolomic approaches. Inhibition of Mir1 by ML316 in respiring yeast diminished mitochondrial oxygen consumption resulting in an unusual metabolic catastrophe marked by citrate accumulation, and death. In a mouse model of azole-resistant oropharyngeal candidiasis, ML316 reduced fungal burden and enhanced azole activity. Targeting Mir1 could provide a new, much needed therapeutic strategy to address the rapidly rising burden of drug-resistant fungal infection.

SIPP, a Novel Mitochondrial Phosphate Carrier, Mediates in Self-Incompatibility.

In Solanaceae, the S-specific interaction between the pistil S-RNase and the pollen S-Locus F-box protein controls self-incompatibility (SI). Although this interaction defines the specificity of the pollen rejection response, the identification of three pistil essential modifier genes unlinked to the S-locus (HT-B, 120K, and NaStEP) unveils a higher degree of complexity in the pollen rejection pathway. We showed previously that NaStEP, a stigma protein with homology with Kunitz-type protease inhibitors, is essential to SI in Nicotiana spp. During pollination, NaStEP is taken up by pollen tubes, where potential interactions with pollen tube proteins might underlie its function. Here, we identified NaSIPP, a mitochondrial protein with phosphate transporter activity, as a novel NaStEP-interacting protein. Coexpression of NaStEP and NaSIPP in pollen tubes showed interaction in the mitochondria, although when expressed alone, NaStEP remains mostly cytosolic, implicating NaSIPP-mediated translocation of NaStEP into the organelle. The NaSIPP transcript is detected specifically in mature pollen of Nicotiana spp.; however, in self-compatible plants, this gene has accumulated mutations, so its coding region is unlikely to produce a functional protein. RNA interference suppression of NaSIPP in Nicotiana spp. pollen grains disrupts the SI by preventing pollen tube inhibition. Taken together, our results are consistent with a model whereby the NaStEP and NaSIPP interaction, in incompatible pollen tubes, might destabilize the mitochondria and contribute to arrest pollen tube growth.

Identification of Reference and Biomarker Proteins in Chlamydomonas reinhardtii Cultured under Different Stress Conditions.

Reference proteins and biomarkers are important for the quantitative evaluation of protein abundance. Chlamydomonas reinhardtii was grown under five stress conditions (dark, cold, heat, salt, and glucose supplementation), and the OD750 and total protein contents were evaluated on days 0, 1, 2, 4, and 6 of culture. Antibodies for 20 candidate proteins were generated, and the protein expression patterns were examined by western blotting. Reference protein(s) for each treatment were identified by calculating the Pearson’s correlation coefficient (PCC) between target protein abundance and total protein content. Histone H3, beta tubulin 1 (TUB-1), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL), and mitochondrial F1F0 ATP synthase subunit 6 (ATPs-6) were the top reference proteins, because they were expressed stably under multiple stress conditions. The average relative-fold change (ARF) value of each protein was calculated to identify biomarkers. Heat shock protein 90B (HSP90B), flagellar associated protein (FAP127) and ATP synthase CF0 A subunit (ATPs-A) were suitable biomarkers for multiple treatments, while receptor of activated protein kinase C1 (RCK1), biotin carboxylase (BCR1), mitochondrial phosphate carrier protein (MPC1), and rubisco large subunit N-methyltransferase (RMT1) were suitable biomarkers for the dark, cold, heat, and glucose treatments, respectively.

Alternate Pathways for Inorganic Phosphate Uptake into Mitochondria

The mitochondrial phosphate carrier (PiC), encoded by the nuclear gene SLC25A3, was purified more than 30 years ago. It is widely believed that PiC serves as the primary means of inorganic phosphate (Pi) uptake into mitochondria for oxidative phosphorylation (oxphos) and for buffering the vast amount of calcium that mitochondria can take up. Yet, the recent discovery of mutations in human PiC, and the development of a PiC floxed mouse only now allow PiC function to be directly studied in vivo. Human SLC25A3 mutations produce a severe clinical phenotype at birth and with striated muscle as a key affected tissue, and mice with cardiac-specific PiC loss eventually develop abnormal cardiac function. Yet, evidence of ample ATP and near normal function despite PiC depletion in humans and mice with PiC depletion suggest that PiC expression far exceeds Pi needs and/or that functional compensation can be substantial. Here we test the hypothesis that Pi enters mitochondria by alternate route(s), allowing substantial mitochondrial ATP production despite near absence of PiC. We generated mice with Tamoxifen(Tam)-inducible skeletal muscle (Skm) PiC knockdown (Tam+Cre+); 3 weeks after Tam, PiC protein was 95% PiC loss; maximal oxphos was ∼35% of control with pyruvate+malate as substrate and rose to ∼50% of control with fatty acid+malate. In contrast, maximal oxphos in Tam+Cre+ mitochondria was abolished with succinate as substrate. Together these observations suggest that, in the context of PiC loss, compensatory Pi uptake may be occur via the dicarboxylate carrier, which exchanges succinate or malate for Pi. Whether this occurs in vivo requires investigation.

Mitochondrial Calcium Uptake and Matrix Calcium Buffering in Skeletal Muscle

Many clues suggest that mitochondrial calcium uptake plays an essential role in muscle function. Mitochondria can contribute to shaping of the sarcoplasmic [Ca2+] transients by their Ca2+ handling and are providing energy for muscle contraction and relaxation by their ATP production. Mitochondrial calcium handling involves uptake, release and Ca2+ chelation in the matrix, which allows storage of vast amounts of Ca2+. Recent identification of the molecular machinery of the Ca2+ uniporter (mtCU) created an opportunity to target specific mechanisms of Ca2+ handling and address their function in Ca2+ homeostasis and contractile function. To elucidate the role of mitochondrial Ca2+ uptake in skeletal muscle (SM) function we ablated MICU1, the Ca2+ sensing regulator of mtCU in mouse. MICU1 ablation in SM resulted in impaired gatekeeping and attenuated Ca2+ uptake and resulted in less endurance exercise performance when challenged with fatigue protocols. However, in this mouse and in cell lines, upon MICU1 ablation protein levels of other components of the mtCU and Ca2+ chelation in the matrix were also altered. To overcome the compensatory effects we decided to apply two strategies. First, we induce acute MICU1 knock out using a tamoxifen-inducible CRE-system. Secondly, we also targeted the mitochondrial phosphate (Pi) carrier (PiC) in a similar manner. Pi transport into mitochondria is crucial for both mitochondrial ATP synthesis and Ca2+ chelation in the matrix. To quantitatively and simultaneously measure Ca2+ uptake and matrix buffering, mitochondria are isolated from SM for fluorometry are loaded with furaFF/AM and are incubated in the presence of rhodFF. SM mitochondria acutely depleted for PiC show a decreased Ca2+ chelation capacity in the matrix and unexpectedly, take up more Ca2+ than the control. When challenged in an incremental exercise test, PiC-ablated mice could only run for 8.5±1.7 min, whereas control mice ran for the full 20 min, showing impaired performance. The effects of acute MICU1 depletion in SM on mtCU composition, mitochondrial Ca2+ handling and SM function are currently evaluated. Thus, our results provide genetic evidence for the contribution of mitochondrial Ca2+ homeostasis to SM function and are expected to determine the function and mechanism dependent on specifically on MICU1 and PiC.

Natural and Induced Mitochondrial Phosphate Carrier Loss: DIFFERENTIAL DEPENDENCE OF MITOCHONDRIAL METABOLISM AND DYNAMICS AND CELL SURVIVAL ON THE EXTENT OF DEPLETION

The relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3. First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of ∼60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ∼85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete medium; however, proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1 or MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (>85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss of function may be independent of phosphate limitation when ATP requirements are not high.

Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate “revertants” viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria.

Novel Mutations in SLC25A3 Encoding the Mitochondrial Phosphate Carrier

It has long been known that the mitochondrial phosphate carrier (PiC), encoded by the nuclear gene SLC25A3, serves a dual role as the primary means of phosphate (Pi) uptake into mitochondria for oxidative phosphorylation and to serve as the main buffering species for calcium entering mitochondria. Yet only lately have mutations in human PiC been identified. We recently described an infant who is a compound heterozygote for two novel PiC variants, c.599T>G (p.Leu200Trp) and c. 886_898delGGTAGCAGTGCTTins CAGATAC (p.Gly296_ Ser300delins GlnIlePro) (Bhoj et al., 2015; PMID 25681081). This compound mutation affects both the muscle-specific isoform, PiC-A, and the ubiquitously expressed isoform, PiC-B. Of note, these two mutations are the first to affect the PiC-B isoform. Phenotypically, the infant initially had severe cardiomyopathy requiring heart transplantation, but had minimal skeletal myopathy and is now relatively healthy. Here, we used patient fibroblasts and yeast expressing humanized versions of Pic1p (the yeast PiC homolog) to analyze these novel PiC variants. Analyses in yeast suggested that the Leu200Trp mutation, affecting a highly conserved leucine, yields functional PiC protein, whereas the del/ins mutation does not. In patient fibroblasts, PiC protein expression was barely detectable, despite the potentially neutral Leu200Trp variant, and phosphorylating oxygen consumption was decreased by ∼50%. The lower oxidative phosphorylation led us to question if mitochondrial fusion activity was altered, since inner mitochondrial membrane fusion depends on mitochondrial ATP. Studies using fibroblasts transfected with mitochondrial matrix-targeted DsRed and photoactivatable GFP indicated suppressed fusion activity of the inner mitochondrial membrane. Overall, these observations provide insight into the pathologic potential of these newly described PiC variants and, more generally, indicate that heterozygosity of a deleterious variant may be sufficient to cause mitochondrial dysfunction and organismal pathology.

Novel mutations in SLC25A3 encoding the mitochondrial phosphate carrier

Novel mutations in SLC25A3 encoding the mitochondrial phosphate carrier

The C Ring of the F1Fo ATP Synthase Forms the Mitochondrial Permeability Transition Pore: A Critical Appraisal.

The C Ring of the F1Fo ATP Synthase Forms the Mitochondrial Permeability Transition Pore: A Critical Appraisal.

Latitudinal variation of freeze tolerance in intertidal marine snails of the genus Melampus (Gastropoda: Ellobiidae).

Abstract Low temperatures limit the poleward distribution of many species such that the expansion of geographic range can only be accomplished via evolutionary innovation. We have tested for physiological differences among closely related species to determine whether their poleward latitudinal ranges are limited by tolerance to cold. We measured lower temperature tolerance (LT50) among a group of intertidal pulmonate snails from six congeneric species and nine locales. Differences in tolerance are placed in the context of a molecular phylogeny based on one mitochondrial (cytochrome oxidase subunit I) and two nuclear (histone 3 and a mitochondrial phosphate carrier protein) markers. Temperate species from two separate lineages had significantly lower measures of LT50 than related tropical species. Range differences within the temperate zone, however, were not explained by LT50. These results show that multiple adaptations to cold and freezing may have enabled range expansions out of the tropics in Melampus. However, northern range limits within temperate species are not governed by cold tolerance alone.

Genetic manipulation of the cardiac mitochondrial phosphate carrier does not affect permeability transition

The Mitochondrial Permeability Transition (MPT) pore is a voltage-sensitive unselective channel known to instigate necrotic cell death during cardiac disease. Recent models suggest that the isomerase cyclophilin D (CypD) regulates the MPT pore by binding to either the F0F1-ATP synthase lateral stalk or the mitochondrial phosphate carrier (PiC). Here we confirm that CypD, through its N-terminus, can directly bind PiC. We then generated cardiac-specific mouse strains overexpressing or with decreased levels of mitochondrial PiC to assess the functionality of such interaction. While PiC overexpression had no observable pathologic phenotype, PiC knockdown resulted in cardiac hypertrophy along with decreased ATP levels. Mitochondria isolated from the hearts of these mouse lines and their respective non-transgenic controls had no divergent phenotype in terms of oxygen consumption and Ca2+-induced MPT, as assessed by swelling and Ca2+-retention measurements. These results provide genetic evidence indicating that the mitochondrial PiC is not a critical component of the MPT pore.

Genetic deletion of the mitochondrial phosphate carrier desensitizes the mitochondrial permeability transition pore and causes cardiomyopathy

The mitochondrial phosphate carrier (PiC) is critical for ATP synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. In addition to its role in energy production, PiC is hypothesized to have a role in cell death as either a component or a regulator of the mitochondrial permeability transition pore (MPTP) complex. Here, we have generated a mouse model with inducible and cardiac-specific deletion of the Slc25a3 gene (PiC protein). Loss of PiC protein did not prevent MPTP opening, suggesting it is not a direct pore-forming component of this complex. However, Slc25a3 deletion in the heart blunted MPTP opening in response to Ca(2+) challenge and led to a greater Ca(2+) uptake capacity. This desensitization of MPTP opening due to loss or reduction in PiC protein attenuated cardiac ischemic-reperfusion injury, as well as partially protected cells in culture from Ca(2+) overload induced death. Intriguingly, deletion of the Slc25a3 gene from the heart long-term resulted in profound hypertrophy with ventricular dilation and depressed cardiac function, all features that reflect the cardiomyopathy observed in humans with mutations in SLC25A3. Together, these results demonstrate that although the PiC is not a direct component of the MPTP, it can regulate its activity, suggesting a novel therapeutic target for reducing necrotic cell death. In addition, mice lacking Slc25a3 in the heart serve as a novel model of metabolic, mitochondrial-driven cardiomyopathy.

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q9; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.