Mitochondrial membrane lipids in the regulation of bioenergetic flux

SUMMARY

Oxidative phosphorylation (OXPHOS) occurs through and across the inner mitochondrial membrane (IMM). Mitochondrial membranes contain a distinct lipid composition, aided by lipid biosynthetic machinery localized in the IMM and class-specific lipid transporters that limit lipid traffic in and out of mitochondria. This unique lipid composition appears to be essential for functions of mitochondria, particularly OXPHOS, by its effects on direct lipid-to-protein interactions, membrane properties, and cristae ultrastructure. This review highlights the biological significance of mitochondrial lipids, with a particular spotlight on the role of lipids in mitochondrial bioenergetics. We describe pathways for the biosynthesis of mitochondrial lipids and provide evidence for their roles in physiology, their implications in human disease, and the mechanisms by which they regulate mitochondrial bioenergetics.

INTRODUCTION

Cellular bioenergetic homeostasis is a tightly regulated balancing act between energy supply and demand, maintained through the tight coupling between the hydrolysis and synthesis of ATP. In most eukaryotic organisms, mitochondria are the predominant supplier of ATP and can contribute >90% of all cellular ATP generation. First proposed by Peter Mitchell’s chemiosmotic theory in the early 1960s,¹ mitochondria contribute heavily to the aerobic synthesis of ATP via oxidative phosphorylation (OXPHOS) and the electron transport chain (ETC), which consists of several oxidation-reduction reactions which “pump” hydrogen ions against their electrochemical gradient from the mitochondrial matrix into the intermembrane space (IMS; Figure 1). This high concentration of protons generates a chemiosmotic potential energy (ΔΨ_m) that, when these protons pass through ATP synthase back into the matrix, ultimately rotates the F₀ and F₁ subunits of ATP synthase to synthesize ATP from ADP and inorganic phosphate. For further details on the structures and functions of the ETC and F₁F₀-ATP synthase, we refer the reader to other reviews.^1–3

Figure 1. Structures of PC, PE, and CL and their distribution in mitochondrial membranes.

Phosphatidylcholine (PC) is a cylindrical, bilayer-forming lipid that is abundant in both outer mitochondrial membranes (OMMs) and inner mitochondrial membranes (IMMs). Phosphatidylethanolamine (PE) and cardiolipin (CL) are both conical, curvature-forming lipids that are more concentrated in cristae. CL, in particular, is almost exclusively localized in the IMM. Structures shown are 16:0/18:1-PC, 16:0/18:2-PE, and 18:2/18:2/18:2/18:2-CL (TLCL).

Mammalian mitochondria contain a unique composition of phospholipids, forming the foundational characteristics of the inner and outer mitochondrial membranes (IMM and OMM), including the formation of ΔΨ_m and aerobic ATP synthesis. The generation and maintenance of ΔΨ_m and the activity of many mitochondrial proteins—namely proteins involved in the ETC and F₁F₀-ATP synthase—depend heavily on the lipid composition of the IMM, as all of these proteins are embedded and operate solely in this subcellular compartment. The distinct lipid makeup of the IMM, including the presence of the mitochondria-specific lipid cardiolipin (CL), appears essential for the efficient functioning of mitochondria, including OXPHOS and many other processes that occur at the IMM or depend upon ΔΨ_m, such as mitochondrial Ca²⁺ uniporter (MCU),⁴ adenine nucleotide translocase (ANT),⁵ nicotinamide nucleotide transhydrogenase (NNT),⁶ and other ion transporters. Nevertheless, we will focus our discussion on the influence of mitochondrial lipids on OXPHOS, with emphases on their biochemical structures and synthesis, pathological conditions related to alterations in the mitochondrial lipidome, and the functional roles these lipid species play in regulating bioenergetics.

LIPID COMPOSITION IN THE MITOCHONDRION

Mitochondria contain two lipid membranes: an OMM and an IMM, separated by the IMS (Figure 1). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and CL comprise 75%–95% of mitochondrial membrane lipids.^7–9 These phospholipids are differentially distributed in the IMM and OMM and serve unique roles in modulating mitochondrial physiology. The OMM encapsulates the outer reticular structure of the mitochondrion and selectively allows the passage of large molecules—such as adenine nucleotides, substrates for OXPHOS, and various proteins. The OMM is composed, in order of abundance, of PC, PE, phosphatidylinositol (PI), and, to a lesser extent, CL, phosphatidic acid (PA), and phosphatidylserine (PS). On the other hand, the mammalian IMM is composed mainly of PE, PC, and CL, which make up nearly 90% of its lipids, with PI and other lipids comprising <10%.¹⁰ Further, the IMM forms a dense network of cristae (lamellar invaginations into the matrix) that separate the IMS from the mitochondrial matrix and anchor proteins that drive bioenergetics—namely mitochondrial ETC complexes I–IV, F₁F₀-ATP synthase, the adenine nucleotide translocator, and uncoupling proteins 1–3—while preserving a tight barrier to maintain ΔΨ_m. The phospholipid composition of the IMM is critical for the formation of cristae folds, which increase the surface area of the IMM and support the generation of a high ΔΨ_m.

PC

PC is the most abundant lipid in mitochondrial membranes, forming ~50% of OMM lipids and ~40% of IMM lipids (compared with 50%–70% of the endoplasmic reticulum [ER] and plasma membranes). PC contains two fatty acids conjugated to a phosphocholine-bound glycerol backbone (Figure 1), which, due to the size of the glycerol-choline head shape and hydrocarbon tails, form a nearly cylindrical shape. This cylindrical structure of PC promotes the self-assembly of lipid bilayers in membranes so that the hydrophobic hydrocarbon chains face each other within the bilayer, whereas the polar headgroups are exposed to the exterior aqueous space surrounding the membrane. Thus, PC is integral in forming semi-permeable mitochondrial bilayers, as required for the proper assembly of β-barrel proteins,¹¹ translocase complexes, and sorting and assembly machinery.¹² Mitochondrial PC likely facilitates OXPHOS,^13,14 as defects in PC biosynthesis promote irregular mitochondrial morphology and lower membrane potential.^15,16

PE

PE, the second-most abundant lipid in the mitochondrial membrane, forms ~30% of the OMM and ~40% of the IMM (compared with 15%–30% of the ER and plasma membranes). PE also comprises two fatty acid chains bound to a glycerol-phosphoethanolamine backbone (Figure 1). However, the polar headgroup of PE is smaller than the choline headgroup of PC. Consequently, PE forms a conical structure and is a non-bilayer component (contributing to curvature) of the mitochondrial membranes, likely contributing to cristae formation.¹⁷ PE is known to bind to complexes I, II, III, and IV of the ETC.^{13,14,18–20}

CL

CL, which is almost exclusively present in mitochondrial membranes, comprises ~5%–15% of the mammalian IMM, depending on the tissue and species,⁷ and a small percentage of the OMM. Approximately 75% of cellular CL is located within the IMM.^21–23 CL is a conical diphosphatidylglycerol containing four fatty acid chains bound to a glycerol backbone (Figure 1).²⁴ Despite the possibility for a high complexity of molecular conformations of CL, select combinations of hydrocarbon chains are more abundantly observed than others, including tetralinoleyl-CL (TLCL).²⁵

CL plays a vital role in cristae folding and mitochondria ultrastructure^26,27 due to its propensity to promote negative membrane curvature.²⁸ CL is known to bind to complexes I, III, IV, and V.^{13,14,18,20,29} CL also supports the assembly of respiratory supercomplexes (see below, subsection mitochondrial electron leak)^26,30–34 and other proteins embedded in the IMM, such as the ANT and uncoupling proteins (see subsection proton uncoupling).^26,35–38 Augmentation or absence of CL induces dramatic phenotypic shifts in mitochondrial physiology, including impaired mitochondrial respiration^26,32 and altered mitochondrial dynamics.^36,39–42 Elamipretide (also known as SS-31)—a compound that has been proposed to directly interact with CL—is thought to stabilize cristae structure and mitochondrial functions.^43–45

PI

PI composes a small fraction of mitochondrial membrane phospholipids (1%–7%, depending on the tissue^8,9,23), most of which are located on the OMM. PI contains two fatty acid chains bound to a glycerol backbone and a phosphoinositol group. PI is ubiquitously present in mammalian cells and plays a role in lipid signaling, vesicular trafficking, and ion channel activity. The removal or masking of the PI derivative, PI(4,5)-bisphosphate, from the OMM causes mitochondrial fragmentation and mitochondrial removal via mitophagy, suggesting that PI is an important factor in the signaling pathways regulating mitochondrial morphology.⁴⁶

Other lipid components of mitochondrial membranes

Mitochondrial membranes consist of several other lipids, including PS, phosphatidylglycerol (PG), PA, lysophospholipids, sterols, and sphingomyelin. These lipids comprise a small fraction of total mitochondrial membrane lipids. The functional roles of most of these lipids in mammalian mitochondria have not been well characterized, although some are critical intermediates in the biosynthesis of other phospholipids. PS (<5% of mammalian mitochondrial membrane phospholipids) is not synthesized in mitochondria but rather at the ER and is imported into mitochondria via ER-OMM membrane contact sites (see subsection lipid import).^8,9,23,47,48 PS is a critical intermediate in providing PE for mammalian mitochondria because the imported PS is converted into PE in the IMM via PS decarboxylase (PSD).

BIOSYNTHESIS AND TRANSPORT OF MITOCHONDRIAL LIPIDS

The IMM is equipped with cellular machinery to generate some membrane lipids, particularly cone-shaped lipids such as PE and CL. Other lipids, such as PC, PS, and PA, are imported from the ER. Below, we describe the processes by which these lipids are synthesized or imported into mitochondria.

Lipid biosynthesis

PC, like PS, is not synthesized in mitochondria. Instead, it is generated at the ER and imported into mitochondria through the mitochondria-associated membranes (MAMs) (Figure 2). Approximately 70% of PC is synthesized by the Kennedy pathway,⁴⁹ where choline undergoes sequential reactions with ATP, cytidine triphosphate (CTP), and diacylglycerol. Alternatively, PC can also be generated by methylation of PE via PE N-methyltransferase (PEMT).^50,51 Some PC can also be produced by the exchange of the serine headgroup of PS with choline via PS synthase 1 (PSS1)^52,53 and by acylation of lysoPC to PC via the Lands cycle and lysoPC acyltransferases (LPCATs).⁵⁴

Figure 2. Mitochondrial lipid biosynthesis and trafficking.

Mitochondrial PC is generated at the ER via the Kennedy pathway and other mechanisms and imported into the OMM by StarD7 (it may also play a role in translocating PC from the OMM to the IMM). PE generated at the ER does not enter mitochondria. Rather, PS generated by PSS1 or PSS2 from PC or PE is imported into mitochondria and further into the IMM by Prelid3b. Mitochondrial PS is then converted to PE via PSD. As a precursor for CL, PA is imported from OMM into IMM by Prelid1. Mitochondrial PA undergoes a series of reactions, including TAMM41, PGS, PTPMT1, and CLS. Nascent CL produced by CLS is transacylated to form TLCL by TAFAZZIN and other de/reacylases. CMP, cytidine monophosphate; Gly, glycerol; Ser, serine; Etn, ethanolamine; Cho, choline.

PE is also synthesized at the ER, but for an unclear reason, PE generated at the ER does not enter mitochondria.⁵⁵ Rather, mitochondrial PE is primarily generated from PS by the PSD localized at the IMM (Figure 2).^17,56 Whole-body knockout of PSD is embryonically lethal in mice, with mitochondria that are fragmented and irregularly dispersed throughout the cell.⁵⁶ As an obligate substrate for PSD, the import of PS from the ER to mitochondria is required for a normal mitochondrial PE level. As described above, ER PS can be generated by PSS1 from PC or from PE by PSS2.⁵⁷ Consistent with this notion, double knockout of PSS1 and PSS2 is also embryonically lethal in mice with similar mitochondrial phenotypes.⁵⁷

CL biosynthesis almost exclusively occurs at IMM (Figure 2). It is generated from PA that is imported into mitochondria from the ER via the Prelid1/TRIAP complex (see below).⁵⁸ PA then undergoes a series of reactions by phosphatidate cytidylyltransferase (TAMM41), PG phosphate synthase (PGS), PG phosphatase (PTPMT1), and CL synthase (CLS).^59,60 Homozygous knockout of PTPMT1 in mice is embryonically lethal and drastically reduces CL and PG levels in mouse embryonic fibroblasts.⁵⁹ PTPMT1 has also been implicated in several types of cancers,^61,62 diabetes,⁶³ and hypertension.^64,65 Four fatty acids on CL generated by CLS often differ in lengths and degrees of desaturation, referred to as nascent CL.⁶⁰ Nascent CL may undergo transacylation to form TLCL (often referred as “mature” CL) by TAFAZZIN (formerly known as TAZ) or by sequential reaction with phospholipase A₂ (PLA₂) and monolyso-CL acyltransferase-1 (MLCLAT1) or acyl-coenzyme A (CoA):lysocardiolipin acyltransferase-1 (ALCAT1, which resides in the ER).^33,66–74 Homozygous knockout of CLS is embryonically lethal in mice.⁷⁵ TAFAZZIN knockdown^76,77 or knockout^78,79 mice develop cardiomyopathy and malformations in mitochondria. TAFAZZIN knockdown mice also exhibit protection from obesity and hepatic steatosis.⁸⁰ Mutations of CLS or TAFAZZIN in humans cause severe and often fatal conditions, most notably cardiomyopathy resulting from impaired mitochondrial respiration.^81,82

Lipid import

Despite initial skepticism surrounding the discovery of mitochondria-ER contact sites in the late 1950s,⁴⁸ MAMs are now recognized as an important nexus for organelle communications.⁸³ 10–30 nm in length, these suborganelle structures allow transient connectivity and inter-organelle crosstalk and exchange of molecules, including lipids.^48,84–87 Emerging evidence describes mechanisms by which lipids are imported into mitochondria via MAM.⁸⁸ In yeast, the vacuolar protein sorting (Vps)13 family of proteins are involved in mitochondrial lipid import.⁸⁹ In humans, VPS13A and VPS13D are localized at MAM, where they similarly facilitate phospholipid import into mitochondria.^89,90 In addition, newly identified mitoguardin (MIGA) and regulator of microtubule dynamics (RMDN) families of proteins appear to exhibit class-specific transport activities toward PS and PA, respectively, translocating them from the ER to the OMM.^91,92

Once lipids arrive at the OMM via MAMs, they must also cross the hydrophobic barrier of the IMS (Figure 2). The mitochondrial contact site and cristae organizing system (MICOS) likely plays a role in phospholipid transfer between the OMM and IMM.⁹³ The MICOS consists of protein complexes that form connections between the OMM and IMM to facilitate cristae junctions.⁹⁴ These structures are essential for IMS lipid and protein transport. Notably, MICOS may rely on CL for proper assembly.^95–98 Phospholipid transfer from the OMM and IMM presumably occurs near MICOS, mediated by class-specific lipid translocators. Steroidogenic acute regulatory protein-related lipid transfer (START)-domain-containing (StarD) family of proteins contain lipid-binding domains implicated in the intracellular lipid transport systems. StarD7-I contains a mitochondria-targeting sequence and likely facilitates the transport of PC from the OMM to the IMM.^99–101 Deletion of StarD7-I decreases mitochondrial PC and impairs mitochondrial respiration.¹⁰² Homozygous knockout of StarD7 is embryonically lethal in mice,^103,104 and human mutations in StarD7 have been associated with familial adult myoclonic epilepsy.¹⁰⁵

PA and PS import is essential for CL and PE biosynthesis, respectively. In both cases, the TP53-regulated inhibitor of apoptosis gene 1 (TRIAP1) and protein of relevant evolutionary and lymphoid interest (PRELI) family of proteins form the TRIAP/PRELID complex to mediate the transfer between the OMM and the IMM. TRIAP (analogous to yeast Mdm35) and PRELI (analogous to yeast Ups) are evolutionarily conserved families of proteins residing in the IMS.^58,106–108 The major isoform of TRIAP in mammals, TRIAP1, forms a protein complex together with either PRELID1, PRELID3a (or SLMO1), or PRELID3b (or SLMO2)¹⁰⁹ and facilitates the selective shuttling of PA (in the case of PRELID1 and PRELID3a) and PS (in the case of PRELID3b) from the OMM to the IMM.⁵⁸ Genetic disruption of the TRIAP/PRELI complex promotes fragmented mitochondria and reduced respiration.⁵⁸ Nucleoside diphosphate kinase D (NDPK-D, also known as NME4) facilitates the transport of CL from the IMM to the OMM.¹¹⁰

GENETIC DISEASES ASSOCIATED WITH MITOCHONDRIAL LIPIDS

A number of human mutations in the genes of mitochondrial lipid biosynthesis have been implicated in conditions with impaired growth and development or premature death (summarized in Table 1). Examples of these conditions include Barth syndrome, which results from impaired CL synthesis⁸²; pathogenic variants for PSD^111–114; chr2-linked familial adult myoclonic epilepsy (FAME2), linked to a mutation in the STARD7 gene¹⁰⁵; Sengers syndrome, caused by defective mutations in acylglycerol kinase (AGK) and PA synthesis^115–117; and Lenz-Majewski syndrome (LMS), caused by gain-of-function mutations in the PS synthesis.^53,118

Table 1.

Summary of known genetic conditions related to mitochondrial lipid biosynthesis in humans

Condition	Affected gene	Lipid primarily affected	Clinical features
Barth syndrome	TAFAZZIN	cardiolipin (TLCL)	cardiomyopathy, neutropenia, blunted cardiac and skeletal muscle growth, low BMI, and adiposity
Pathogenic variants of PSD	PISD	phosphatidylethanolamine	congenital cataracts, skeletal dysplasia, and white matter changes
Mutations in STARD7	STARD7	unclear	myoclonic tremors and generalized tonic-clonic seizures
Sengers syndrome	AGK	phosphatidic acid, cardiolipin	congenital cataracts, hypertrophic cardiomyopathy, skeletal muscle weakness, and advanced exercise-induced lactic acidosis
Lenz-Majewski syndrome	PTDSS1	phosphatidylserine, phosphatidylethanolamine	sclerosing bone dysplasia, intellectual disability, distinct craniofacial, dental, cutaneous, and distal limb abnormalities

Barth syndrome

Barth syndrome is an X-linked, potentially fatal condition characterized by pediatric onset of cardiomyopathy, neutropenia, blunted cardiac and skeletal muscle growth, and exercise tolerance caused by mutations in the TAFAZZIN gene.⁸² Over 70% of infants with Barth syndrome develop cardiomyopathy within their first year of life, with 14% requiring heart transplantation.¹¹⁹ Many, but not all, patients with Barth syndrome present with low BMI and low adiposity.¹²⁰ In humans, ~80% of CL in the heart is normally composed of TLCL,^121,122 suggesting that cardiac function might rely on this species of CL. As observed in patients with Barth syndrome, mutations in the TAFAZZIN gene caused a 75% loss of TLCL and an increase in MLCL,¹²³ with few alterations to other mitochondrial phospholipid species (i.e., PC, PE, PS, etc.).⁷¹ There is evidence that loss of CL remodeling disrupts the spatial organization of IMM proteins.¹²⁴ A recent study using adeno-associated virus serotype 9 (AAV-9) vectors as a means of TAFAZZIN gene replacement in a mouse model of Barth syndrome showed promise as a potential therapeutic approach.⁷⁸

Pathogenic variants of PSD

Mutations in PISD, a gene that encodes the PSD enzyme, have been identified to cause mitochondrial disease that clinically presents with congenital cataracts, skeletal dysplasia, and white matter changes.^112,114 In one study, two female siblings were found to have compound heterozygous mutations (one missense and one splice variant) on the PISD gene. Fibroblasts derived from both sisters contained low levels of mitochondrial PE and exhibited decreased mitochondrial respiration coupled with impaired complex IV activity, lower ΔΨ_m, and increased mitochondrial mass compared with fibroblasts from control subjects.^112,114

Other genetic conditions potentially influenced by mitochondrial lipids

Mutation in the first intron of the STARD7 gene has been implicated in FAME2.¹⁰⁵ This condition is characterized by myoclonic tremors and generalized tonic-clonic seizures. Proton magnetic resonance spectroscopy shows increased levels of choline in the cerebellum of individuals with FAME2,¹²⁵ an observation that may be predicted with dysfunctional STARD7. Nevertheless, the mutation does not appear to alter STARD7 expression in patient-derived skin fibroblasts. Thus, the relevance of mitochondrial lipids in FAME2 remains speculative.

Sengers syndrome is a rare autosomal recessive disorder characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal muscle weakness, and advanced exercise-induced lactic acidosis.¹¹⁷ Sengers syndrome is caused by loss-of-function mutations in the AGK gene.^115,116 Homozygous mutations in the AGK gene result in a more severe form of Sengers syndrome that results in early infant mortality. Although AGK functions in a major biosynthetic pathway for PA (a necessary precursor to CL), the hypothesis that patients with Sengers syndrome exhibit alterations in mitochondrial membrane phospholipid composition has not been investigated in detail. AGK binds with the TIM22 protein import complex, which plays a role in the molecular import and assembly of various proteins, including ATP synthase.¹²⁶ In cells, while global knockout of AGK resulted in mitochondrial defect, mutating the catalytic site of AGK did not recapitulate these phenotypes. Thus, Sengers syndrome may be caused by the activity of AGK independent of its effect on mitochondrial membrane lipid composition. The development of an in vivo model of Sengers syndrome is needed to confirm these findings.

LMS is clinically characterized by the compilation of sclerosing bone dysplasia, intellectual disability, and distinct craniofacial, dental, cutaneous, and distal limb abnormalities.¹¹⁸ Whole-genome sequencing of LMS patients suggested that the condition is caused by SNPs constellated around the gene encoding the PSS1 enzyme (PTDSS1).⁵³ All study participants exhibited heterozygous missense, gain-of-function mutations in the PTDSS1 gene.^127–129 Indeed, fibroblasts obtained from patients with LMS exhibited a higher rate of PS synthesis compared with that in control fibroblasts, indicating that overproduction of PS via PSS1 is likely the primary defect underlying LMS.⁵³ Phenotypes associated with LMS are likely contributed by the pleiotropic role of PS, including, but not limited to, the function of PS in mitochondria.

NON-COMMUNICABLE CONDITIONS ASSOCIATED WITH MITOCHONDRIAL LIPIDS

Alterations in mitochondrial membrane lipids have also been implicated in non-communicable conditions and diseases such as exercise, metabolic-dysfunction-associated steatotic liver disease (MASLD), diabetes, and neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.

Exercise

Exercise training is known to promote mitochondrial biogenesis in skeletal muscle.^130,131 We have shown that this adaptation coincides with a remodeling of mitochondrial lipid composition.¹³² A greater exercise capacity, either with treadmill exercise training or in rats bred for running capacity,¹³³ is associated with an increase in the proportion of PE in mitochondria. Conversely, physical activity induced by hindlimb unloading or reduced cage size lowers PE content in mitochondria.^132,134 These changes in mitochondrial PE are likely driven by changes in PSD expression that also coincide with exercise. Loss of mitochondrial PE in skeletal muscle substantially reduces skeletal muscle contractility, supported by reduced capacities for respiration and ATP production, as well as an increase in mitochondrial electron leak that promotes oxidative stress.¹³²

MASLD

Alterations in mitochondrial bioenergetics have been implicated in the pathogenesis of MASLD,¹³⁵ but the underlying mechanisms for these changes are unclear. In mice, an increase in hepatic mitochondrial CL is observed early in obesity but decreases with the progression to MASLD.¹³⁶ Mice with hepatocyte-specific deletion of CLS exhibit diet-induced steatohepatitis, though whether this effect is mediated by the functions of CL on OXPHOS is unknown.¹³⁷ In contrast, mice with global deletion of TAFAZZIN are protected from MASLD, though this is confounded by their hypermetabolic phenotype.⁸⁰ In rats, increased peroxidized CL has been observed with MASLD.^136,138 Exogenous CL, but not peroxidized CL or other phospholipids, restored some of the OXPHOS defects associated with MASLD.^137,139 How CL mediates bioenergetic alterations induced by MASLD remains to be fully elucidated.

Diabetes

In both type 1 and type 2 diabetes, the reduced insulin secretory response in pancreatic β cells contributes to hyperglycemia. Particularly for type 2 diabetes, changes in mitochondrial energetics have been implicated in the pathogenesis of defective glucose-stimulated insulin secretion.¹⁴⁰ Mitochondrial CL may play a role in this process. In global TAFAZZIN knockdown mice, loss of TAFAZZIN promotes a ~50% reduction in insulin secretion measured ex vivo.¹⁴¹ Likewise, the global deletion of phospholipase A₂β, one of the alternative enzymes for CL transacylation, makes β cells defective for insulin secretion.¹⁴² Tissue-specific gain- or loss-of-function studies for these enzymes are needed to confirm that these changes are due to the lack of CL in the β cells.

Neurodegenerative disease

Alterations to mitochondrial membrane phospholipids, particularly CL, may contribute to the development of Alzheimer’s disease and Parkinson’s disease. The brain appears to have a greater diversity of CL species compared with other tissues, with up to 100 different CL species reported.^143,144 CL is particularly perturbed by the accumulation of tau proteins in models of Alzheimer’s disease^145,146 and α-synuclein (αS) in models of Parkinson’s disease.^147–153 In the early stages of Alzheimer’s disease, synaptic, but not non-synaptic, mitochondria exhibit a decreased abundance of CL (specifically TLCL) and an increased abundance of PC and lyso-PC.¹⁴⁶ These alterations are accompanied by a decrease in the activity of complex I and lower ATP concentrations in neurons. Tau proteins appear to bind with CL directly, causing mitochondria to swell and release cytochrome c.¹⁴⁵ Nonyl acridine orange, a molecule known to bind to CL,¹⁵⁴ prevented tau-induced mitochondrial damage, suggesting a causal tau-CL mechanism in the pathology of Alzheimer’s disease. Similarly, αS perturbs mitochondrial lipid membranes by interacting with membranes containing greater curvature¹⁴⁸ and acidic lipids¹⁵⁵ (such as CL), with a higher affinity for unsaturated acyl chains. The high affinity for αS-CL binding appears to reduce complex I activity in synaptic mitochondria.¹⁵⁶

Influence of cell types on mitochondrial membrane lipid composition

The composition of mitochondrial membranes differs between tissues in the same organism. In particular, the diversity of CL species is dramatically different between tissues in the same organism, potentially reflecting mitochondria adapted for biological processes that are specific to the cell type.¹⁵⁷ TLCL content in mouse heart, liver, brain, and gastrocnemius muscle can range from 7.1% (brain) to 80.6% (liver) of total CL.^158,159 It is not entirely clear why TLCL content is comparatively high in liver mitochondria, where its respiratory capacity is not particularly high.¹⁶⁰ It is possible that they represent a potential requirement for TLCL in gluconeogenesis, lipogenesis, and/or transamination, which experience high flux in hepatocytes. CL is also more highly represented in the mitochondrial lipidome of cardiomyocytes, which maintain constant, high energetic demands. Alternatively, tissues with lower ATP demand, such as adipose, contain relatively low levels of CL.

INFLUENCE OF MITOCHONDRIAL LIPIDS ON BIOENERGETICS

As evidenced by the examples of human mutations in the genes of mitochondrial lipid biosynthesis, loss of these lipids likely induces defects in mitochondrial bioenergetics that contribute to their pathology. Membrane lipids likely influence OXPHOS through complex mechanisms. These mechanisms include lipid-to-protein binding that affects enzyme activity, membrane properties that affect lateral diffusion of electron carriers in IMM as well as the conductance of ions across IMM, and cristae architecture influencing suborganellar compartmentalization (Figure 3). Advances in structural biology and access to mitochondrial bioenergetic phenotyping platforms, such as high-resolution respirometry, fluorometry, electrophysiology, and microscopy, have made it possible to more deeply study the influence of mitochondrial membrane lipids on energy transduction through OXPHOS.^161–166

Figure 3. Diverse roles of mitochondrial phospholipids regulating OXPHOS.

The effects of IMM lipids on bioenergetics are likely mediated by lipid-to-protein interactions that affect OXPHOS enzyme activity, membrane properties that influence lateral diffusability of electron carriers through IMM and proton conductance across the IMM, by influencing the interaction of respiratory complexes to facilitate efficient electron transfer and regulate electron leak, and cristae architecture that regulates compartmentalization of mitochondrial membrane potential.

ATP production

Respiration (oxygen consumption rate [OCR] or O₂ flux [JO₂]) is often measured to assess energy flux through OXPHOS. Although useful to study, the OCR is just one of the metrics for OXPHOS activity. The ETC harnesses the reduction potential between NADH (or succinate) and molecular O₂ to drive the movement of electrons from complex I/II to III and to IV, while coupling the electron transfer to proton pumping from the matrix to the IMS to generate membrane potential. Oxygen can be consumed either at the terminal complex IV (4H⁺ + 4e⁻ + O₂ → 2H₂O) or in a small fraction as a result of premature electron leak (e⁻ + O₂ → O₂⁻). Thus, oxygen consumption is approximately, but not exactly, equimolar to energy influx to OXPHOS. In many cases, respiration is a reasonable estimation of the mitochondria’s ability to take part in ATP synthesis. Nonetheless, ATP production may be decoupled by a number of mechanisms (electron leak, proton leak, etc.), such that the direct measurement of the rate of ATP synthesis is desirable when assessing the energy output of OXPHOS.

Evidence suggests that mitochondrial CL, PE, and PC are all essential for OXPHOS. These lipids are known to directly interact with complex I–V to affect their activities.^{13,14,18–20,36} Mutations in TAFAZZIN resulting in Barth syndrome, which reduces TLCL, diminishes respiratory capacity in skeletal muscle mitochondria in these patients.¹⁶⁷ Genetic ablations that negatively influence the enzymes of CL biosynthesis almost universally reduce oxygen consumption or ATP production across tissues.^32,168–170 However, in the liver and in brown adipose tissue (BAT), loss of CL has no effect on ATP production.^80,171,172 Similarly, loss of mitochondrial PE reduces oxygen consumption and ATP production in skeletal muscle,^132,173 but not in BAT.¹⁷¹ It is highly intriguing that the depletion of mitochondrial lipids in different cell types promotes differential bioenergetic phenotypes. These differences are likely partly driven by the differential proteome and lipidome in these cells, as well as their differential metabolic demands. For example, locomotor activity commands a robust component of bioenergetic demands in skeletal muscle, necessitating exceptionally high ATP flux. In contrast, proton uncoupling (see below) induced by thermogenic demands primarily drives bioenergetic demands in BAT, requiring lower allocation of membrane potential energy for ATP synthesis (also discussed in the subsection influence of cell types on mitochondrial membrane lipid composition above).

Proton uncoupling

Uncoupling protein 1 (UCP1), which resides in the IMM, is largely responsible for thermogenesis in brown and beige adipocytes. UCP1 uncouples the mitochondrial membrane potential (ΔΨ_m) to ATP synthesis by translocating protons in IMS back into the matrix independent of complex V.^174,175 This process effectively uncouples the ETC from ATP production, and the energy from the ΔΨ_m is dissipated as heat.

Brown and beige adipocytes are highly responsive to ambient temperature and regulate thermogenesis by modulating UCP1 transcription and activity. Membrane lipids appear to play an important role in this regulation, as CL tightly binds to UCP1.¹⁷⁶ Cold exposure in mice induces a robust, time-dependent increase in the expression of enzymes involved in CL synthesis and transacylation.¹⁷⁷ Loss- or gain-of-function studies show that CL positively regulates thermogenesis.^171,177 However, bioenergetic phenotyping of brown adipose mitochondria isolated from CLS-deleted mice show normal UCP1-dependent respiration despite substantially compromised thermogenesis.¹⁷¹ Thus, while it is clear that CL is essential for thermogenesis, the exact mechanism by which it regulates UCP1 needs further clarification. In contrast, mitochondrial PE appears essential for UCP1-dependent respiration and proton conductance in brown adipocytes.¹⁷¹ Mitochondrial PE also robustly responds to ambient temperature. It is unknown whether PE directly binds to UCP1, but there are some reports that PE increases the protonophoric activity of UCP1.¹⁷⁸

Mitochondrial electron leak

Electrons donated from NADH or succinate can prematurely leak to molecular O₂ or other acceptors prior to doing so in a highly controlled manner in complex IV. For example, electron stalling in the Q-pool (reduced and oxidized mixture of coenzyme Q) is thought to lead to reverse electron transfer in complex I, enabling electrons to reduce O₂ into O₂⁻ (oxygen radical) that in turn reacts with water to become H₂O₂. Excessive production of these oxidants may induce oxidative stress and they may also have important signaling roles. Similar to proton uncoupling, mitochondrial electron leak (or reduced electron transfer efficiency) can increase respiration without channeling its energy for ATP synthesis.

An increase in mitochondrial electron leak has been implicated in the pathogenesis of Barth syndrome. Some studies report an increase in mitochondrial electron leak with TAFAZZIN deficiency.^168,179 On the other hand, in a study where mitochondrial electron leak was quantified from 11 potential sites (including those in the ETC and substrate catabolism), TAFAZZIN deficiency did not alter electron transfer efficiency in any of them.¹⁸⁰ Indeed, neutralizing mitochondrial H₂O₂ by overexpression of mitochondria-targeted catalase did not ameliorate cardioskeletal myopathy in TAFAZZIN knockdown mice.¹⁶⁸ Homozygous, but not heterozygous, deletion of PSD promoted mitochondrial electron leak in skeletal muscle,^132,173 suggesting that a robust decrease in mitochondrial PE is required to reduce electron transfer efficiency. In BAT, neither deleting CLS nor PSD had an effect in increasing mitochondrial electron leak.¹⁷¹ We speculate that brown adipocytes are particularly resistant to an increase in mitochondrial electron leak due to the strong bioenergetic “pull” (energetic demand) induced by UCP1.

In mammals, up to 80%–85% of individual mitochondrial OXPHOS enzymes may be found in clusters, known as “supercomplexes” (or respirasomes).^181,182 There remains some controversy regarding whether these supercomplexes represent the cause or consequence of efficient electron transfer. A recent study suggests that respirasomes are not required for maintaining normal bioenergetics.¹⁸³ Other studies suggest that supercomplex assembly contributes directly to IMM curvature.¹⁸⁴ Nevertheless, respiratory complexes appear to either permanently or transiently become close in proximity, such that the probability of electron leaking from the ETC is reduced. Lymphoblasts from patients with Barth syndrome have decreased amounts of the CI₁CIII₂ supercomplex.¹⁸⁵ Likewise, mice with cardiomyocyte-specific TAFAZZIN knockout¹⁸⁶ and inducible TAFAZZIN knockdown^33,187 demonstrate lower levels of supercomplexes, suggesting that mature CL is necessary for higher-order respirasome formation. Similarly, loss of mitochondrial PE also reduces supercomplex formation in mouse muscle and in Chinese hamster ovary (CHO) cells.^17,132

Cristae architecture

Mitochondrial cristae folds are a hallmark of mitochondrial morphology and are critical to OXPHOS. ETC, UCP1, and ATP synthase are all localized in the cristae.^188–191 It is known that individual cristae can become biochemically separated from the rest of IMS and other cristae units by closing the cristae junction, forming cristae vesicles (Figure 4). As the two major cone-shaped lipids abundant in IMM, PE and CL are presumably highly concentrated in cristae. The concentration of these membrane lipids would be predicted to directly influence membrane curvature and, thus, the shape and volume of the cristae vesicles.

Figure 4. How phospholipids might influence membrane potential by altering cristae volume.

Cone-shaped phospholipids such as PE and CL create the negative curvature found in cristae. Increased abundance of these lipids (left) makes it possible for cristae to make more acute turns, resulting in smaller cristae volume. The electrochemical concentration gradient of IMM would be directly influenced by the volume of cristae vesicles, as fewer protons would be predicted to be needed to achieve the same concentration.

Why is the shape and volume of cristae vesicles important for bioenergetics? This is because membrane potential is not merely a mathematical sum of protons pumped by ETC; rather, it is that sum divided by the volume of IMS. In other words, ΔΨ_m is an electrochemical concentration gradient across IMM. Thus, changes in the IMS volume would be predicted to directly influence ΔΨ_m. We subscribe to the idea that each cristae vesicle can become segregated from other cristae vesicles and the rest of IMS (inner boundary membrane [IBM]) through the closing of the cristae junction (Figure 4), such that each cristae vesicle possesses distinct ΔΨ_m (heteropotential mitochondria).^192–195 In this model, ΔΨ_m in any given mitochondrion is not uniform, giving rise to electrochemically distinct ATP-synthesizing units. If this were the case, the lipid composition of the cristae membrane would tremendously impact ΔΨ_m through its effect on cristae vesicle volume. Cone-shaped lipids such as PE and CL should induce membrane curvature and reduce the volume of cristae vesicles, in turn amplifying the unit of ΔΨ_m gained per unit of ETC flux. Consistent with this notion, mitochondria deficient in PTPMT1 (one of the enzymes of CL biosynthesis) produce cristae vesicles that are bigger in size.¹⁶⁵ Mitochondria from patients with Barth syndrome, animal models of TAFAZZIN, and CLS deficiencies have fewer cristae folds.^{76,78,171,186} Similar morphological changes are observed with impairment in PSD in humans and in mouse models.^{17,56,132,171} Another important consideration is how the bioenergetic consequence of cristae segregation is relevant to mitochondrial dynamics, as phospholipids are also known to influence these processes.⁴⁰

How morphologically dynamic are individual cristae, and how does membrane lipid composition potentially influence ΔΨ_m? Considering the heteropotentiality and highly dynamic nature of mitochondrial cristae architecture, it is, perhaps, logical to postulate that there exists a synergistic system by which ΔΨ_m and lipid composition of individual cristae co-regulate each other to dynamically remodel cristae size and shape, similar to the plasma membrane.^196,197 In this model, which we term the “bagpipe hypothesis” (Figure 5), it is possible that the architecture of individual mitochondrial cristae is dynamically responsive to the magnitude of the difference in electric charge across the IMM, having a potential to temporarily diffuse its pressure by expanding cristae volume (“ballooning”). In more elastic membranes, such an effect would be immediately counteracted by the compression forces from the IMM that promote efflux back into the matrix. Thus, the framework of this bagpipe hypothesis would stipulate that the size and volume of the individual cristae folds are tightly regulated by a ΔΨ_m and/or lipid composition feedback system, which dictates cristae architecture, ATP production, and proton leak. Interestingly, compared with cylindrical lipids such as PC, PE is likely to increase the elasticity of biological membranes due to the higher bending rigidity of PE.¹⁹⁸ Because this may be a property of conical lipids, it is also likely that CL increases the elasticity of biological membranes. Therefore, loss of PE and CL could increase the flaccid properties of the IMM, allowing greater expansion capacity. Further, this could be the primary reason that TLCL is deemed the main “functional” form of CL, as the saturation, number of acyl chains,¹⁹⁹ or Ca²⁺ ion bonding¹²² in CL could considerably influence the structural, and thus the elastic, properties of the IMM.

Figure 5. The bagpipe hypothesis.

Bagpipes are played when air travels through the chanter that produces the melody, as well as through the drones that produce continuous sound. The continuous sound from the chanter and drones are enabled by air blown into the blowstick and, importantly, by compression forces to the bag. Our hypothesis proposes that mitochondrial cristae function similarly. Protons are pumped into the IMS via the ETC. As protons accumulate in the IMS, this produces an expansion force (ΔΨ), which is opposed by the IMM. The IMM membrane composition influences the elastic properties of the membrane. Highly rigid membranes retain their shape better and thus are able to generate higher membrane potentials with fewer protons in the IMS. Membranes with low rigidity are more likely to expand with a greater osmotic pressure in the IMS, stabilizing the thermodynamic gradient between the matrix and the IMS. This expansion of the IMM then requires a higher proton concentration in the IMS to drive ATP synthesis, or protons escape via various mechanisms.

How might this dynamic role of mitochondrial membrane lipids serve as a direct influence on mitochondrial bioenergetics? In membranes with greater compression forces, perhaps such as those with high CL and PE, membrane expansion is limited by the more rigid membranes. Because of this, cristae volume is also limited. Therefore, a lower proton influx into the IMS facilitated by the ETC is required to meet the ΔΨ_m needed for ATP production (~60 mV). On the other hand, as the proton motive force driving ATP synthesis is dictated by ΔΨ_m, which is influenced by cristae volume, a more compliant IMM would increase overall cristae volume, thus maintaining the thermodynamic state of the IMS. However, we might also factor in the effect that membrane expansion has on the ETC. It could be the case that, as the IMM expands, so does the distance between proteins embedded in the IMM. The expansion would then serve a dual purpose in slowing the rate of electron transfer between ETC complexes, thus generating an immediate self-regulating feedback mechanism where cristae expansion slows down H⁺ accumulation in the cristae, thus maintaining a relatively constant ΔΨ_m. The increased diffusion distance would also have implications for increased reactive oxygen species (ROS) production, as the impaired rate of electron transfer would block electron transfer between ETC proteins and carriers, increasing the likelihood that some electrons would be released from their bound chaperones. Although this thought experiment is intriguing, it would need to be thoroughly tested. Recent advancements in ultra-resolution real-time in situ imaging techniques (such as stimulated emission depletion microscopy) could provide avenues to understand the dynamic changes that occur in mitochondrial cristae in response to bioenergetic flux.

CONCLUSIONS AND PERSPECTIVES

Human mutations in genes that encode the enzymes of mitochondrial membrane biosynthesis are associated with OXPHOS dysfunction, which is detrimental to health. OXPHOS occurs through and across these IMM lipids, making them an integral component to the energy-transducing processes that yield ATP synthesis. The effects of these lipids are likely mediated by lipid-to-protein interaction that affects enzyme activity, membrane properties that influence lateral diffusability of electron carriers and proton conductance, and cristae architecture that regulates compartmentalization of mitochondrial membrane potential. Furthermore, lipids influence bioenergetics in cell-type-specific manner. In cardiac and skeletal myocytes where ATP production is the primary demand for OXPHOS, mitochondrial lipids exquisitely regulate ATP synthesis. In UCP1-positive adipocytes where there are other purposes for OXPHOS, mitochondrial lipids do not appear to strongly influence ATP synthesis. We speculate that a mitochondrial microenvironment (proteomic, metabolomic, and bioenergetic milieu) that is specific to the cell likely interacts with membrane lipids to ultimately give rise to its OXPHOS phenotype.

Recent emergence in lipid mass spectrometry, combined with tools in cell biology and mitochondrial phenotyping, enabled a greater understanding of the diverse and fundamental roles that the mitochondrial membrane phospholipids play in regulating bioenergetics. We believe these mechanisms to play an important role in how mitochondria adapt to altering cellular energetic demands and that defects in these processes may contribute to diseases associated with chronic energy inbalance. We identify two areas of research that will have the greatest impact in advancing this field. One is in structural biology to characterize the biophysical nature of the interaction of these lipids with respiratory complexes. Results from bioenergetic phenotyping could be used to guide targeted questions on interactions that are most likely to be relevant. Advances in techniques to obtain high-resolution localization of lipids is another area. Current technology does not allow suborganellar visualization of lipids, as these molecules largely lack reliable probes and spatial resolution of mass spectrometry is currently insufficient to resolve organelles. How are the membrane lipids localized in the cristae and are they clustered to form microdomain-like rafts to facilitate spatial distribution of respirasomes? The molecular resolution of these processes will likely provide the greatest insights into the functional significance of these lipids in regulating mitochondrial bioenergetics.