New Insights on the Uptake and Trafficking of Coenzyme Q

Coenzyme Q (CoQ) is an essential lipid with many cellular functions, such as electron transport for cellular respiration, antioxidant protection, redox homeostasis, and ferroptosis suppression. Deficiencies in CoQ due to aging, genetic disease, or medication can be ameliorated by high-dose supplementation. As such, an understanding of the uptake and transport of CoQ may inform methods of clinical use and identify how to better treat deficiency. Here, we review what is known about the cellular uptake and intracellular distribution of CoQ from yeast, mammalian cell culture, and rodent models, as well as its absorption at the organism level. We discuss the use of these model organisms to probe the mechanisms of uptake and distribution. The literature indicates that CoQ uptake and distribution are multifaceted processes likely to have redundancies in its transport, utilizing the endomembrane system and newly identified proteins that function as lipid transporters. Impairment of the trafficking of either endogenous or exogenous CoQ exerts profound effects on metabolism and stress response. This review also highlights significant gaps in our knowledge of how CoQ is distributed within the cell and suggests future directions of research to better understand this process.


Introduction
Coenzyme Q (CoQ), or ubiquinone, is a vital lipid needed for generation of energy and a crucial antioxidant that preserves membrane structure and function. Although it is synthesized within the mitochondria of eukaryotes, it is present in all cellular membranes and is also a component of lipoproteins. CoQ 10 (the isoform synthesized by humans and most mammals) is an enormously popular supplement and a medically important therapeutic. Yet, its insolubility in water means it must be trafficked between membranes via lipid vesicles, micelles, chaperones, or transport proteins. The pathways and mechanisms that move this lipid from one membrane to another are still largely undefined. Elucidating the pathways of this trafficking will aid our understanding of the functional roles of CoQ and may lead to improved therapeutics.
The rescue of CoQ 10 -deficient animals supplemented with synthetic hydrophilic CoQ precursors or "bypass therapies" indicates that bioavailability is a key limitation of CoQ 10 supplementation [6]. Bypass therapy for patients with primary CoQ deficiency is currently being studied as a potential alternative to supplementation with CoQ 10 [47,48]. In bypass therapies, analogs of 4-HB are used as alternate ring precursors that function to "bypass" a blocked step in the CoQ biosynthetic pathway. For example, 2,4-dihydroxy-benzoic acid (2, partially restores CoQ biosynthesis in mice harboring mutations in Coq7 (also termed Mclk1) [49]. Mice with certain mutations of Coq9 also respond to 2,4-diHB treatment [50]. However, 2,4-diHB also inhibits the endogenous CoQ biosynthetic pathway, possibly due to competition with natural substrates of the COQ enzymes upstream of the Coq7 hydroxylation step [49]. Bypass of human COQ6 mutations using vanillic acid, a methoxylated analog of 4-HB, also has efficacy in a cultured cell model [51]. Due to the lack of a hydrophobic polyisoprenyl tail, the analogs of 4-HB are significantly more bioavailable than CoQ 10 , and the restoration of endogenous biosynthesis may achieve a more effective rescue [52].

The S. cerevisiae Model
S. cerevisiae (hereafter referred to as yeast) is a single-celled fungus that has been widely studied as a simple, eukaryotic model organism. Yeast cells are capable of both fermentation and aerobic respiration, and they divide rapidly through a budding process in which smaller daughter cells bud off from a larger mother cell. These cells are approximately five microns in diameter, between bacterial and human cells in size. Yeast cells can readily switch between haploid a and haploid α cells and mate to form diploid cells, which can further sporulate to produce four haploids [53]. Many eukaryotic cellular processes have been first identified and extensively characterized in yeast, including cell cycle regulation, vesicle trafficking, protein secretion, and autophagy. Many of these processes are highly conserved between yeast and humans. In fact, 47% of the 414 essential genes in S. cerevisiae with a 1:1 human ortholog can be functionally replaced by the corresponding human ortholog [54]. These well-characterized pathways, ease of genetic manipulation, and strong conservation between yeast and humans make yeast a powerful model organism.
Much work has been performed to elucidate the biosynthetic pathway and understand the cellular function of CoQ 6 in yeast. The COQ1-COQ11 genes encode mitochondrially targeted polypeptides that are required for efficient CoQ 6 biosynthesis [37][38][39]. Strains with a deletion or deleterious mutation in one of the COQ1-COQ9 genes, termed coq mutants, are unable to synthesize CoQ 6 ( Figure 3a). These coq mutants are thus incapable of cellular respiration and fail to grow in a non-fermentable medium. In most cases, expression of the human COQ ortholog in the respective yeast coq mutant rescues efficient CoQ 6 biosynthesis and restores growth in a non-fermentable medium, highlighting the strong conservation of CoQ biosynthesis from yeast to humans. In addition to the presence of all the Coq polypeptides, efficient CoQ 6 synthesis requires Coq3-Coq9 and Coq11 to form a mega-complex on the matrix side of the inner mitochondrial membrane, termed the CoQ synthome [38,39]. The formation of this complex is analogous to the Complex Q assembly that occurs in human cells [39]. The strong conservation of CoQ biosynthesis and many functions from yeast to humans suggest a conserved pathway of CoQ transport. Yeast coq mutants are rescued with exogenous CoQ6 but not non-isoprenoid CoQ analogues. (a) CoQ6 was not detected in any of the coqΔ mutants. CoQ6 (pmol/mg protein) was determined based on a CoQ6 standard curve as described in . (b) Each of the coq mutants, from coq3Δ to coq9Δ, accumulated early CoQ6 intermediates (HHB and HAB). The amount of HHB and HAB was calculated as the area of the MS peak/mg protein. (c) Yeast mutants harboring deletions in either the COQ1 or COQ2 gene showed more robust rescue in response to exogenous CoQ6 treatment than do the other single coq null mutants (coq3Δ-coq9Δ). (d) Additional deletions of either COQ1 or COQ2 restored the deficient CoQ6-rescue of a coq3Δ mutant, but do not affect the phenotype observed in coq1Δ or coqΔ2 strains. Columns represent the degree of rescue (in %) ± SD of a strain compared to WT, which is defined as 100% and represented as a dashed line. Statistically significant differences between a specific coqΔ mutant and one of its counterparts (another coqΔ mutant) are denoted with numbers in parentheses on top of the columns. (1) represents differences comparing to coq1Δ, (2) represents differences comparing to coq2Δ, (3) represents differences comparing to coq3Δ, etc. Three or more independent rescue experiments were performed for every strain. Asterisks on top of the columns represent significant differences when compared to WT (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). (e) Structures of CoQ analogues. Panels (a-d) reproduced with permission from Fernandez-del-Rio et al. Free Radicals in Biology and Medicine, published by Elsevier, 2020 [18]. Created with Biorender.com (Accessed on 27 June 2023). Yeast coq mutants are rescued with exogenous CoQ 6 but not non-isoprenoid CoQ analogues. (a) CoQ 6 was not detected in any of the coq∆ mutants. CoQ 6 (pmol/mg protein) was determined based on a CoQ 6 standard curve as described in . (b) Each of the coq mutants, from coq3∆ to coq9∆, accumulated early CoQ 6 intermediates (HHB and HAB). The amount of HHB and HAB was calculated as the area of the MS peak/mg protein. (c) Yeast mutants harboring deletions in either the COQ1 or COQ2 gene showed more robust rescue in response to exogenous CoQ 6 treatment than do the other single coq null mutants (coq3∆-coq9∆). (d) Additional deletions of either COQ1 or COQ2 restored the deficient CoQ 6 -rescue of a coq3∆ mutant, but do not affect the phenotype observed in coq1∆ or coq∆2 strains. Columns represent the degree of rescue (in %) ± SD of a strain compared to WT, which is defined as 100% and represented as a dashed line. Statistically significant differences between a specific coq∆ mutant and one of its counterparts (another coq∆ mutant) are denoted with numbers in parentheses on top of the columns. (1) represents differences comparing to coq1∆, (2) represents differences comparing to coq2∆, (3) represents differences comparing to coq3∆, etc. Three or more independent rescue experiments were performed for every strain. Asterisks on top of the columns represent significant differences when compared to WT (* p < 0.05, ** p < 0.01, *** p < 0.001). (e) Structures of CoQ analogues. Panels (a-d) reproduced with permission from Fernandez-del-Rio et al. Free Radicals in Biology and Medicine, published by Elsevier, 2020 [18]. Created with Biorender.com (Accessed on 27 June 2023).

Growth Rescue of coq Mutants in a Non-Fermentable Medium Supplemented with Exogenous CoQ 6
As previously stated, coq mutants are unable to synthesize CoQ 6 and are thus respiratory deficient and fail to grow in a medium containing a non-fermentable carbon source (Figure 3a). Deletion of COQ1 or COQ2 that are involved in early steps of CoQ 6 biosynthesis prevents the accumulation of early CoQ 6 intermediates, 3-hexaprenyl-4-hydroxybenzoic acid (HHB) and 3-hexaprenyl-4-aminobenzoic acid (HAB), while deletion of any one of the COQ3-COQ9 genes that are required for later biosynthetic steps accumulate these early CoQ intermediates (Figure 3b). Significantly, direct supplementation of exogenous CoQ 6 restores respiration and growth of coq mutants [55,56], though the degree of rescue depends on the nature of the coq mutant. When supplemented with exogenous CoQ 6 in non-fermentable media, coq1∆ and coq2∆ single mutants exhibit wild-type or near wild-type levels of growth, while each of the coq3∆-coq9∆ single mutants exhibit growth that is significantly lower than wild-type cells (Figure 3c). Deletion of either COQ1 or COQ2 in the coq3∆ single mutant restores rescue to that of wild-type cells or to the coq1∆ and coq2∆ single mutants (Figure 3d), indicating that the accumulation of early CoQ 6 intermediates, HHB and HAB, impairs growth rescue of coq3∆-coq9∆ mutants [18]. It is possible that HHB/HAB act as detergents or may interfere with membrane trafficking steps needed for transit of exogenously added CoQ 6 . CoQ 6 uptake is favored by the presence of peptone in the culture media [57]. The digested proteins in peptone likely bind CoQ 6 and enable its entry into the aqueous compartment of endocytic vesicles. Following supplementation with exogenous CoQ 6 , the coq mutants also show a significant delay in growth compared to wild-type cells as they make the diauxic shift from fermentation to respiration [57]. Supplementation with either hydrophilic CoQ isoforms that can spontaneously diffuse across non-continuous membranes (CoQ 2 , CoQ 4 ), or hydrophobic CoQ isoforms that cannot diffuse (CoQ 6 ) are both capable of rescuing growth. Moreover, supplementation with CoQ 2 can bypass defects in endocytosis and membrane trafficking [19,58]. The ability of yeast to uptake exogenous CoQ, transport it to the mitochondria, and in turn restore respiration in coq mutants makes yeast a powerful model organism to investigate CoQ uptake and transport.
Studies with supplementation of non-isoprenoid CoQ analogs indicate that the isoprenoid tail is essential for efficient uptake and growth rescue of coq mutants in a nonfermentable medium. Idebenone, decylQ, and MitoQ are three such analogs containing a ten-carbon alkyl tail ( Figure 3e). The tail end of idebenone and MitoQ are further modified with a hydroxyl group or triphenylphosphonium moiety, respectively, with the latter exhibiting mitochondrial targeting. None of these three analogs were able to restore growth of coq mutants when supplemented in a non-fermentable medium [58]. Although decylQ and idebenone restore respiration in isolated mitochondria, it is speculated that mitochondrial accumulation of these analogs in whole cells is insufficient to support growth. Conversely, MitoQ accumulates in mitochondria of whole cells, but is poorly oxidized by Complex III and thus unable to restore respiration [58]. In fact, supplementation with high concentrations of decylQ inhibits growth rescue of coq mutants with CoQ 2 , perhaps through competition with an unidentified CoQ-binding protein [19]. Supplementation assays with analogs of CoQ 2 where the prenyl tail has been modified show that each of the analogs tested are capable of restoring respiration in isolated mitochondria, but the presence of the double bond between C2 and C3 in the first isoprenoid unit of exogenously supplied CoQ is essential for efficient uptake in whole cells and growth in a non-fermentable medium [19]. Taken together, the decylQ impairment of growth rescue by CoQ 2 and the structural requirement in the isoprenoid tail of exogenous CoQ suggest the presence of an essential protein that can distinguish subtle differences in CoQ structure for efficient delivery or retention of exogenous CoQ to mitochondrial respiratory complexes.
It is possible that the yeast Coq10 polypeptide may fulfill this role of a CoQ chaperone. Yeast Coq10 is peripherally associated with the matrix side of the inner mitochondrial membrane, but is not part of the endocytic pathway, the respiratory complexes, or the CoQ synthome [59]. The yeast Coq10 polypeptide contains a steroidogenic acute regulatory-related lipid transfer (START) domain, which binds CoQ n isoforms of variable tail lengths and analogs of late-stage CoQ-intermediates such as DMQ 3 [60]. Yeast Coq10 is needed for respiratory function, and may be involved in transport of newly synthesized CoQ 6 to Complex III [61]. COQ10A and COQ10B are human orthologs of yeast Coq10, and partially rescue both the respiratory defect and sensitivity to lipid peroxidation of the coq10∆ yeast mutant [62]. Human STARD7 is structurally related to the yeast Coq10 and human COQ10 polypeptides, binds CoQ 4 in vitro, and plays a role in vivo in transporting mitochondrial CoQ 10 to the plasma membrane to suppress ferroptosis [63]. Human saposin B binds CoQ 10 , and is postulated to function as a transporter [64,65]. Mammalian CoQ transport proteins are discussed in Section 3.1.5. It is likely that many other CoQ transporter or chaperone proteins remain to be discovered.  [19]. Sixteen such genes were found by screening a library of~4800 ORF∆coq∆ double mutants. The deletion of these ORFs resulted in impaired growth rescue on a medium containing a non-fermentable carbon source when supplemented with exogenous CoQ 2 or CoQ 4 . However, it is important to note that CoQ 2 and CoQ 4 have shorter isoprenoid tails than CoQ 6 , passively diffuse across noncontinuous membranes, and move independently of lipid trafficking mechanisms [18,19].

Identification of Essential Genes in
To further examine the role of these ORFs as well as other candidates in the uptake and trafficking of CoQ 6 , Fernández-Del-Río et al. tested whether exogenous CoQ 6 restored growth in a screen of 40 ORF∆coq2∆ double mutants in a non-fermentable medium [18]. This group of 40 genes included the 16 ORF∆coq2∆ double mutants identified by James et al. [19]. The rescue assay compared the ORF∆coq2∆ double mutant to the coq2∆ single mutant, and to the ORF∆ single mutant in a non-fermentable medium when supplemented with exogenous CoQ 6 [18]. The degree of growth rescue in a medium supplemented with exogenous CoQ 6 was shown to be dependent on the distinct COQ gene that was disrupted (see Section 2.1). Six ORF∆coq2∆ double mutants demonstrated reduced growth in a non-fermentable medium with exogenous CoQ 6 as compared to either the coq2∆ or the ORF∆ single mutants, suggesting a role for these ORFs in CoQ 6 uptake and transport to the mitochondria. Fernández-Del-Río et al. reasoned that supplementation with the hydrophilic isoform CoQ 2 should be able to circumvent the trafficking defect observed with CoQ 6 in these mutants [18]. Indeed, the growth rescue afforded by CoQ 2 supplementation in these ORF∆coq2∆ double mutants far exceeded that of CoQ 6 . Such growth rescue assays of ORF∆coq2∆ double mutants provide a powerful tool to identify genes that are required for the uptake of exogenous CoQ 6 and its trafficking to mitochondria. However, more work is needed to unravel the role these gene products play in uptake and transport of exogenous CoQ 6 .
Interestingly, two genes, RTS1 and CDC10, were identified as essential in both screens ( Figure 4) [18,19]. Rts1 is a regulatory subunit of protein phosphatase 2A (PP2A), which plays a role in regulation of cell size control, the mitotic spindle orientation checkpoint, and septin ring organization and disassembly during cytokinesis [66,67]. It is possible that Rts1 in complex with PP2A plays a regulatory role in CoQ 6 uptake and transport. CDC10 is one of four core septins in yeast, a group of GTPases that assembles into filaments and higher order structures [68]. Septin filaments assemble into a septin ring at the bud neck in yeast and act as a scaffold to recruit other proteins to drive cytokinesis [69]. Septins have been shown to interact with a subset of endocytic proteins [70], and SEPT9, the human homolog of Cdc10, has been implicated in endosomal trafficking [71]. More work is needed to determine if the role of Cdc10 in CoQ 6 uptake and transport is involved in endocytosis or another septin function. [77]. This is curious because trafficking between the plasma membrane and mitochondria is reported to occur via clathrin-mediated endocytosis [78]. However, trafficking of proteins and lipids to mitochondria is likely mediated by extensive contact sites between mitochondria and other intracellular membranes ( Figure 4) [79,80]. Fernández-Del-Rio et al. screened ORFΔcoq2Δ double mutants predicted to impair selected membrane contact sites such as the endoplasmic reticulum-mitochondria encounter structure (ERMES), the nucleus-vacuole junction (NVJ), and the vacuole-mitochondria patch (vCLAMP) [18]. The four other essential genes for uptake and trafficking of exogenous CoQ 6 to respiratory complexes identified by Fernández-Del-Río et al. are RVS161, RVS167, NAT3, and VPS1 ( Figure 4) [18]. Rvs161 and Rvs167 are amphiphysin-like membrane proteins that form a heterologous dimer and promote membrane curvature [72]. Rvs161 and Rvs167 interact with several other proteins, including Vps1, at the plasma membrane for the vesicle scission step of endocytosis [73]. Vps1 is a GTPase involved in membrane fusion and fission. Vps1 also operates in other cellular processes, including vacuolar protein sorting, actin cytoskeleton organization, late Golgi retention of proteins, and peroxisome biogenesis [74]. Nat3 is a catalytic subunit of the NatB complex that mediates acetylation of the N-terminus of approximately 20% of proteins in yeast and humans [75,76]. The role that each of these genes plays in CoQ 6 uptake and trafficking remains unclear.
Typically, mitochondria are excluded from schemes depicting endosomal trafficking [77]. This is curious because trafficking between the plasma membrane and mitochondria is reported to occur via clathrin-mediated endocytosis [78]. However, trafficking of proteins and lipids to mitochondria is likely mediated by extensive contact sites between mitochondria and other intracellular membranes ( Figure 4) [79,80]. Fernández-Del-Rio et al. screened ORF∆coq2∆ double mutants predicted to impair selected membrane contact sites such as the endoplasmic reticulum-mitochondria encounter structure (ERMES), the nucleus-vacuole junction (NVJ), and the vacuole-mitochondria patch (vCLAMP) [18].
However, none of these ORF∆coq2∆ double mutants displayed impaired growth rescue when treated with exogenous CoQ 6 . These results could suggest that CoQ 6 transport may occur independently of these membrane contact sites, or that transport through contact sites is redundant. For example, mitochondria dependence on phospholipid exchange has been shown to depend on either ERMES or vCLAMP [81]. Loss of ERMES stimulates vCLAMP and vice versa; however, loss of both is lethal [81]. Yeast genes that are redundant or essential for growth on a non-fermentable carbon source will not be detected by such exogenous CoQ 6 growth-rescue screens. It is also possible that other lipid transport mechanisms operate independently of both endocytic and contact site-mediated trafficking.

Rescue of coq Mutants with Exogenous CoQ Is Variable and Depends on the Genetic Background of the Yeast Strain
Growth rescue of coq mutants in a non-fermentable medium with exogenous CoQ 6 is dependent on the yeast genetic background. For example, the commonly used BY4741/BY4742 laboratory yeast strain takes up and transports exogenous CoQ 6 to the mitochondrial respiratory complexes, and was used in the screen to detect trafficking defects (Section 2.2.1) [18]. This is also the case for the W303-1B and CEN.PK2-1C yeast genetic backgrounds [82]. Yet, certain yeast strains harboring a coq7∆ null allele, such as the EG103coq7∆ or the FY250coq7∆ mutant yeast strains, fail to be rescued when the growth medium is supplemented with exogenous CoQ 6 . The content of CoQ 6 in the gradient-purified mitochondrial fraction of these strains was only 35% and 8% of the content present in the mitochondria isolated from the EG103 and FY250 wild-type parental strains, respectively. However, the CoQ 6 content in the plasma membrane fraction isolated from EG103coq7∆ and the FY250coq7∆ strains exceeded or equaled the content of CoQ 6 normally present in the plasma membrane fraction of the parental strains [82]. In these mutant strains, the plasma membrane redox activity was augmented, suggesting that the exogenous CoQ 6 present in the plasma membrane was functional [82]. Thus, it is likely that such strains unable to be rescued for growth by exogenous CoQ 6 likely have a defect in trafficking the CoQ 6 from the plasma membrane to the mitochondria [82]. Both the parental EG103 and the EG103coq7∆ mutant strains were shown to secrete carboxy-peptidase Y and accumulate small vacuoles, markers that characterize impaired membrane trafficking [57]. It would be important to test whether these mutants are rescued by supplementation with exogenous CoQ 2 , since this analog does not depend on lipid trafficking.
To test the idea that defects in endocytosis or endomembrane trafficking alter CoQ 6 uptake and transport to the mitochondria, Padilla-López et al. interrogated ORF∆coq3∆ double mutants whose ORF deletion, such as ERG2, PEP12, TLG2, or VPS45, disrupts several endocytic transport steps [57]. Supplementation with exogenous CoQ 6 failed to rescue growth of these ORF∆coq3∆ double mutants in a non-fermentable medium, suggesting that the endocytic and endomembrane trafficking pathways are involved in CoQ 6 uptake and transport. Subcellular fractionation confirmed that each of these ORF∆coq3∆ double mutants have decreased uptake of exogenous CoQ 6 in mitochondria, Golgi, vacuole, and plasma membrane compared to WT and coq3∆ controls, further illustrating the impact of disrupting these trafficking pathways in CoQ 6 uptake. Moreover, the low CoQ 6 content in the plasma membrane of these mutants suggests that most of the exogenously supplied CoQ 6 cannot be directly incorporated into the plasma membrane.
It must be noted that both coq7∆ and coq3∆ mutants accumulate HHB and HAB, early CoQ 6 intermediates that act to impair growth rescue in a non-fermentable medium with exogenous CoQ 6 . Indeed, Fernández-Del-Río et al. showed that these ORF∆coq3∆ double mutants had significantly less robust growth in a non-fermentable medium when compared to the ORF∆coq2∆ double mutant counterpart (Figure 3c,d) [18]. More work must be performed in yeast to further determine the role of endocytosis and endomembrane trafficking in CoQ 6 uptake and transport in yeast models. Previous work in the human promyelocytic HL-60 cell line has shown that both endogenously synthesized CoQ 10 and exogenously supplied CoQ 9 are distributed across membranes through the brefeldin Asensitive endo-exocytic pathway (see Section 3.1.1) [83]; however, such experiments have not been performed in yeast.

CoQ 6 Transport to and from the Mitochondria
It is likely that uptake and trafficking of exogenous and endogenous CoQ 6 share common pathways. Recent studies suggest that the ERMES may play a role in both CoQ 6 biosynthesis and distribution. ERMES is a tethering complex that forms at the membrane contact site between the ER and the outer mitochondrial membrane and is composed of two mitochondrial subunits embedded in the outer membrane (Mdm10 and Mdm34), an ER-localized subunit (Mmm1), and a cytosolic subunit (Mdm12) (Figure 4) [84]. The four subunits are functionally linked to phospholipid biosynthesis and calcium signaling [84]. Recent structural analyses show that ERMES forms bridge-like structures between the ER and mitochondrial outer membrane, consistent with providing a conduit for phospholipid transport [85]. The mitochondrial contact site and cristae organization system (MICOS), a mega complex that regulates cristae junction organization, has been shown to assemble near ERMES sites, and ERMES function is required for MICOS assembly [86]. The Mmm1-Mdm12 complex has been shown to mediate phospholipid transfer in vitro and mutations in MMM1 and MDM12 lead to impaired lipid transfer through ERMES in vivo [87].
Intriguingly, Coq polypeptides that are components of the CoQ synthome at the inner mitochondrial membrane are visualized as puncta or CoQ domains that lie adjacent to ERMES puncta at the outer mitochondrial membrane [88,89]. Disruption of ERMES decreases the number of CoQ domains [88,89]. Moreover, ERMES mutants show increased whole-cell accumulation of CoQ 6 and CoQ 6 -intermediates but decreased content of CoQ 6 in isolated mitochondria, suggesting that CoQ 6 and CoQ 6 -intermediates accumulate in non-mitochondrial membranes [89]. Current observations suggest that destabilization of the CoQ synthome results in reduced sequestration of CoQ 6 and CoQ 6 intermediates in the mitochondria; alternatively, disruption of ERMES might lead to enhanced stability of non-mitochondrial CoQ 6 [89]. Despite this role for ERMES in CoQ domain formation and CoQ 6 production and distribution, direct transport of CoQ 6 through ERMES has not yet been observed. Moreover, none of the four ERMES genes were identified as essential for CoQ uptake and transport in ORF∆coq2∆ double mutants [18]. However, disruption of ERMES results in an expansion of vCLAMP contact sites, and deletion of both contacts has been shown to be lethal [81]. Thus, disruption of ERMES might increase compensatory contact sites to counteract potential defects in CoQ 6 trafficking.
The idea that other membrane contact sites can compensate for lipid transport defects in ERMES mutants has been reinforced by recent structure/function studies of the VPS13 family of proteins. Yeast Vps13 is a 360 kDa protein composed of an extensive beta-sheet or "taco shell" lined by hydrophobic residues that can form a conduit for phospholipid transfer between organelles [90]. Yeast Vps13, Vps39, and Mcp1 form a mitochondria-vacuole contact site (Figure 4), and are required for the survival of yeast ERMES mutants [91]. Mcp1 is a mitochondrial outer membrane phospholipid scramblase, and partners with the Vps13 lipid bridge protein to re-equilibrate lipids as they are either inserted into or removed from the cytosolic leaflet of acceptor or donor membranes [92]. Vps39 is the vacuole fusion factor responsible for vCLAMP contact sites [81]. It is not yet known whether these bridge-like phospholipid transporters strategically positioned at contact sites also move neutral lipids, such as CoQ.
Recent work has identified yeast mitochondrial Cqd1 and Cqd2 proteins that influence cellular distribution of endogenous CoQ 6 [93]. Intriguingly, both are inner membrane proteins that face the intermembrane space, and potentially provide a connection for transfer of CoQ 6 from the inner membrane to the outer membrane and the ER ( Figure 4). Disruption of CQD1 or CQD2 either diminishes or enhances the mitochondrial CoQ 6 content, respectively, without altering overall cellular CoQ 6 content [93]. Cqd1 and Cqd2 are members of the UbiB family of atypical kinases/ATPases. Coq8, an essential protein for CoQ 6 biosynthesis and part of the CoQ synthome, is also a member of the UbiB family whose ATPase activity may be coupled to the extraction of hydrophobic CoQ 6 intermediates from the inner membrane for subsequent processing by membrane-associated Coq polypeptides in the matrix [94]. Point mutations of both Cqd1 and Cqd2 at conserved protein-kinase-like (PKL) or UbiB motif residues disrupted their individual impact on CoQ 6 distribution, indicating that Cqd1 and Cqd2 activities rely on atypical kinase/ATPase activity, though further biochemical work is needed to prove enzymatic activity. Furthermore, haploinsufficiency of the CQD1 human ortholog ADCK2 is reported to cause aberrant mitochondrial lipid oxidation and myopathy associated with CoQ 10 deficiency [95].
Cqd1 participates in a contact site between outer and inner mitochondrial membranes (Figure 4), and overexpression of Cqd1 and Cqd2 elicits contact sites between the ER and mitochondria [96]. Taken in conjunction with the effect of Cqd1 on CoQ 6 retention within mitochondria, and the effect of Cqd2 on promoting CoQ 6 exit from mitochondria, these proteins likely play a role in CoQ 6 trafficking to and from the mitochondria in yeast. It would be interesting to determine whether the Cqd1 complex is co-localized with the CoQ synthome and ERMES, as it provides the "missing link" between these two. Interestingly, deletion of both Cqd1 and Cqd2 in yeast restores the normal distribution of yeast CoQ 6 [93], indicating that other trafficking mechanisms must operate. Hence, it is likely that other CoQ 6 transporters contribute to trafficking.
Distribution of CoQ 6 between mitochondrial and non-mitochondrial membranes plays important functional roles in mitochondrial respiration, response to oxidative stress, and redox control. The cqd1∆ single mutant has decreased mitochondrial CoQ 6 content, grows slowly in a medium containing glycerol as the sole non-fermentable carbon source, has increased non-mitochondrial CoQ 6 content, and shows enhanced resistance to treatment with PUFAs [93]. Conversely, the cqd2∆ single mutant has increased mitochondrial CoQ 6 content, normal growth on a medium containing glycerol, and is more sensitive to treatment with PUFAs [93]. The trafficking of CoQ to non-mitochondrial membranes (including the plasma membrane) mediates resistance to ferroptosis, a type of cell death due to lipid peroxidation [32,33]. CoQ 6 in the plasma membrane functions as a potent antioxidant and co-antioxidant (Figure 1b), and is also in the PMRS (Figure 1c). In respiratory deficient cells (yeast and HL-60 cultured cells), there is an increased content of CoQ at the plasma membrane [97,98]. This recruitment of CoQ to the PMRS enables cytosolic NADH to be oxidized to NAD + , together with export of the electrons to extracellular acceptors, such as ascorbate, and thereby maintains the ratio of cytosolic NAD + /NADH [99,100]. Hence, the pathways that control the trafficking of both endogenous and exogenous CoQ exert profound effects on metabolism and stress response.

Mammalian Cell Culture Models
Mammalian cell culture has been integral in the study of the uptake and distribution of CoQ for many decades. Among the reasons for using cell culture(s) to study CoQ uptake and trafficking are the high degree of specificity with which one can control their environment, the ease and accuracy of administration of nutrients, hormones, and in this case CoQ, the decreased physiological variability as compared to live animals. However, whether cells in culture accurately reflect their true physiological context is often difficult to determine.
In addition to uptake and trafficking of exogenous CoQ, cell culture has been used to study the CoQ synthome and endogenous CoQ biosynthesis [101]. The functional roles of CoQH 2 as an antioxidant have also been characterized in culture [102,103], with a specific emphasis on plasma membrane-localized CoQ as a protectant against ferroptosis [32,33,104,105]. Cells in culture have likewise proven to be a powerful model for the study of CoQ uptake and distribution. In the following section, we discuss CoQ uptake and intracellular trafficking in epithelial, endothelial, blood, dermal, and HeLa cells. Subsequently, the supplementation and efficacy of CoQ 10 H 2 and CoQ 10 will be compared, before concluding with a discussion about cell receptors that mediate uptake of exogenous CoQ 10 .

Epithelial Cell Lines
Orally supplemented CoQ 10 is first taken up by the epithelial cells of the digestive tract [2,9,106]. However, the mechanism by which enterocytes absorb CoQ 10 is not well understood. Several reviews and studies state that the uptake of CoQ 10 occurs by a simple process of passive diffusion [2,9]. A combination of passive diffusion and receptormediated uptake is responsible for absorption of fatty acids [107], vitamin E [108], and cholesterol [109] in enterocytes. Niemann-Pick C1-like 1 (NPC1L1) plays an important role in uptake of dietary cholesterol; its disruption in mice results in a 50% decreased cholesterol absorption [109]. Recent studies suggest that membrane protein transporters are also important for the uptake of CoQ 10 . The human intestinal epithelial cell line CaCo-2 provides a model for much of the recent research on CoQ absorption in the digestive tract. CaCo-2 is a colorectal adenocarcinoma cell line that is used to study transport across the intestinal epithelium, including the absorption of various hormones and small molecules such as insulin [110], anthocyanin [111], and calcitonin [112]. Among the advantages of this cell line is its formation of tight junctions, microvilli that form a brush border on the luminal or apical side, and transport proteins that allow the uptake and digestion or transport of nutrients to the basolateral side, all of which can simulate the in vivo digestion of molecules via oral supplementation ( Figure 5) [113]. study the CoQ synthome and endogenous CoQ biosynthesis [101]. The functional roles of CoQH2 as an antioxidant have also been characterized in culture [102,103], with a specific emphasis on plasma membrane-localized CoQ as a protectant against ferroptosis [32,33,104,105]. Cells in culture have likewise proven to be a powerful model for the study of CoQ uptake and distribution. In the following section, we discuss CoQ uptake and intracellular trafficking in epithelial, endothelial, blood, dermal, and HeLa cells. Subsequently, the supplementation and efficacy of CoQ10H2 and CoQ10 will be compared, before concluding with a discussion about cell receptors that mediate uptake of exogenous CoQ10.

Epithelial Cell Lines
Orally supplemented CoQ10 is first taken up by the epithelial cells of the digestive tract [2,9,106]. However, the mechanism by which enterocytes absorb CoQ10 is not well understood. Several reviews and studies state that the uptake of CoQ10 occurs by a simple process of passive diffusion [2,9]. A combination of passive diffusion and receptor-mediated uptake is responsible for absorption of fatty acids [107], vitamin E [108], and cholesterol [109] in enterocytes. Niemann-Pick C1-like 1 (NPC1L1) plays an important role in uptake of dietary cholesterol; its disruption in mice results in a 50% decreased cholesterol absorption [109]. Recent studies suggest that membrane protein transporters are also important for the uptake of CoQ10. The human intestinal epithelial cell line CaCo-2 provides a model for much of the recent research on CoQ absorption in the digestive tract. CaCo-2 is a colorectal adenocarcinoma cell line that is used to study transport across the intestinal epithelium, including the absorption of various hormones and small molecules such as insulin [110], anthocyanin [111], and calcitonin [112]. Among the advantages of this cell line is its formation of tight junctions, microvilli that form a brush border on the luminal or apical side, and transport proteins that allow the uptake and digestion or transport of nutrients to the basolateral side, all of which can simulate the in vivo digestion of molecules via oral supplementation ( Figure 5) [113].  CaCo-2 cells can assimilate solubilized CoQ 10 from digested oral supplements, such as CoQ 10 in liposomes and micelles. Supplementation of CaCo-2 cells with CoQ 10 -infused liposomes altered the mRNA levels of 694 genes involved in signaling, metabolism, and general transport [106]. Many of these proteins are directly regulated by CoQ, such as Caspase-3, which is inhibited by CoQ 10 at the plasma membrane [114]. CoQ 10 uptake by CaCo-2 cells is enhanced when solubilized forms of CoQ 10 were administered via simulated digestive micellization [115]. Eight different commercial CoQ 10 products were mixed with fat-free yogurt and olive oil, and subjected to gastric conditions by treatment with hydrochloric acid and porcine pepsin. Following this treatment, lipase, bile extract, and porcine pancreatin were added to simulate digestion. This digestion micellized the CoQ 10 , which was then extracted from the aqueous layer of the resultant mixture. Of the eight CoQ 10 products, four exhibited greater than 60% micellization in the aqueous layer: γ-Cyclodextrin complexed-CoQ 10 tablet, liposomal CoQ 10 solution, solubilized CoQ 10 capsule, and a γ-Cyclodextrin/solubilized CoQ 10 powder termed Hydro-Q-Sorb ® [115]. γ-Cyclodextrin is a water-soluble octasaccharide of particular interest to the optimization of CoQ uptake [116]. The sugar features eight glucose subunits interlinked to form a cylindrical shape, characterized by its hydrophilic exterior and hydrophobic interior, which can encapsulate CoQ 10 . γ-Cyclodextrin is one of many formulations of CoQ 10 that have been assessed for their ability to enhance its bioavailability [117]. The liposomal CoQ 10 and solubilized CoQ 10 displayed the greatest rate of micellization, but it was Hydro-Q-Sorb ® that was absorbed most by CaCo-2 cells. Hydro-Q-Sorb ® exhibited a dramatic increase in CoQ 10 content after a four-hour incubation when compared to incubation with the non-solubilized CoQ 10 powder reference, despite Hydro-Q-Sorb ® containing only 20% of the concentration of CoQ 10 as the reference powder [115]. The correlation between CoQ 10 product micellization and CaCo-2 uptake was weak, suggesting that other factors regulate CoQ 10 uptake besides micellar transport [115]. Regardless, there does exist a positive relation between the micellization of CoQ 10 and its internalization in the CaCo-2 cell model, indicating the importance of lipid dispersion in CoQ uptake.
The CaCo-2 human intestinal epithelial cell model has been used to compare the relative effects of CoQ 10 and CoQ 10 H 2 uptake, as several studies postulate that CoQ 10 H 2 is more bioavailable than CoQ 10 [118]. Supplementation with CoQ 10 H 2 poses a challenge because the hydroquinone undergoes rapid autoxidation under cell culturing conditions [119]. As such, studies designed to compare treatment with CoQ 10 H 2 versus CoQ 10 should monitor autoxidation of the CoQ 10 H 2 during incubation conditions. Recently, Naguib et al. [120] found that CoQ 10 H 2 complexed with γ-cyclodextrin remained stable in an aqueous salt solution for up to seven days, with the majority of the original CoQ 10 H 2 not undergoing autoxidation to CoQ 10 , demonstrating that CoQ 10 H 2 can be supplemented with high purity in this form. Failla et al. [118] investigated the relative uptake and transport of CoQ 10 and CoQ 10 H 2 across the CaCo-2 monolayer using a previously described simulated digestion method [115]. CoQ 10 or CoQ 10 H 2 was subjected to "digestion" and added to the apical medium. Following a four-hour incubation, the total cell-associated CoQ 10 content and the total amount of CoQ 10 transported into the basolateral medium were determined. Both apical uptake into CaCo-2 cells, and transepithelial transport was significantly higher for the micellized CoQ 10 H 2 as compared to the micellized CoQ 10 [118]. The authors showed that incorporation of CoQ 10 H 2 into micelles during the digestion process was twice as efficient when compared with CoQ 10 [118]. Non-complexed CoQ 10 has been shown to have very low permeability across the CaCo-2 monolayer [118,121], indicating that in the absence of CoQ 10 incorporation into micelles or liposomes, transport across the brush border is extremely inefficient. Thus, both physiological and pharmaceutical solubilization methods for CoQ 10 (or CoQ 10 H 2 ) are needed to enhance its absorption across the small intestine epithelium.
Small intestine epithelial cells express NPC1L1, a transport protein involved in cholesterol and vitamin E transport and metabolism (Figure 6a) [108,122,123]. Recent work has identified a role for NPC1L1 in the intestinal absorption of CoQ. NPC1L1 was overexpressed in Madin-Darby canine kidney (MDCK) epithelial cells to assess NPC1L1-mediated CoQ 9 and CoQ 10 transport [124]. MDCK cells are epithelial cells derived from kidney tissue of a cocker spaniel. NPC1L1 was expressed over 50-fold in human small intestine and duodenum compared to the kidney, making this mutant MDCK model reasonably analogous to that of intestinal CoQ uptake [125]. CoQ 10 and CoQ 9 uptake was found to increase significantly in NPC1L1-overexpressed MDCK cells, as compared to control MDCK cells. Furthermore, treatment with ezetimibe, a selective inhibitor of NPC1L1, decreased CoQ 10 and CoQ 9 uptake of NPC1L1-overexpressed cells back to control levels, implicating NPC1L1 as a CoQ-transport protein [124]. This study demonstrates the exis-tence of intestine-specific proteins that function to increase CoQ 10 absorption, among other lipids. However, the mechanism of NPC1L1-CoQ 10 interaction remains unclear.
kidney tissue of a cocker spaniel. NPC1L1 was expressed over 50-fold in human small intestine and duodenum compared to the kidney, making this mutant MDCK model reasonably analogous to that of intestinal CoQ uptake [125]. CoQ10 and CoQ9 uptake was found to increase significantly in NPC1L1-overexpressed MDCK cells, as compared to control MDCK cells. Furthermore, treatment with ezetimibe, a selective inhibitor of NPC1L1, decreased CoQ10 and CoQ9 uptake of NPC1L1-overexpressed cells back to control levels, implicating NPC1L1 as a CoQ-transport protein [124]. This study demonstrates the existence of intestine-specific proteins that function to increase CoQ10 absorption, among other lipids. However, the mechanism of NPC1L1-CoQ10 interaction remains unclear. (a) Exogenous CoQ uptake and intracellular CoQ distribution are mediated by several known mechanisms in mammalian cell culture. CD36 and NPC1L1 recognize and import exogenous CoQ within brown adipose tissue and small intestine epithelial cells, respectively. The endomembrane system facilitates intracellular trafficking of CoQ between membrane-bound vesicles. Dual-localized STARD7 in the mitochondria and cytosol support endogenous CoQ biosynthesis and distribution to the plasma membrane, respectively. (b) Model of lipoprotein-associated CoQ trafficking at the blood-brain barrier. SR-B1 and RAGE regulate influx of the HDL-associated CoQ across the bloodbrain barrier, while LRP-1 regulates the efflux of LDL-associated CoQ. 1, receptor-mediated endocytosis; 2, recycling to apical side via the same receptor; 3, transcytosis to basolateral side; 4, transport to lysosome; 5, recycling or efflux of CoQ to apical membrane via LDL receptor family proteins; 6, paracellular transport detected due to defects in tight junctions. HDL, high-density lipoprotein; LDL, low-density lipoprotein. Created with BioRender.com (Accessed on 28 June 2023).
The HepG2 liver epithelial cell line has played an important role in the development of new strategies for exogenous CoQ10 uptake. Moschetti et al. [126,127] engineered a nanodisk composed of a phosphatidylcholine bilayer circumscribed by apolipoprotein- (a) Exogenous CoQ uptake and intracellular CoQ distribution are mediated by several known mechanisms in mammalian cell culture. CD36 and NPC1L1 recognize and import exogenous CoQ within brown adipose tissue and small intestine epithelial cells, respectively. The endomembrane system facilitates intracellular trafficking of CoQ between membrane-bound vesicles. Dual-localized STARD7 in the mitochondria and cytosol support endogenous CoQ biosynthesis and distribution to the plasma membrane, respectively. (b) Model of lipoprotein-associated CoQ trafficking at the bloodbrain barrier. SR-B1 and RAGE regulate influx of the HDL-associated CoQ across the blood-brain barrier, while LRP-1 regulates the efflux of LDL-associated CoQ. 1, receptor-mediated endocytosis; 2, recycling to apical side via the same receptor; 3, transcytosis to basolateral side; 4, transport to lysosome; 5, recycling or efflux of CoQ to apical membrane via LDL receptor family proteins; 6, paracellular transport detected due to defects in tight junctions. HDL, high-density lipoprotein; LDL, low-density lipoprotein. Created with BioRender.com (Accessed on 28 June 2023).
The HepG2 liver epithelial cell line has played an important role in the development of new strategies for exogenous CoQ 10 uptake. Moschetti et al. [126,127] engineered a nanodisk composed of a phosphatidylcholine bilayer circumscribed by apolipoprotein-A1, where CoQ 10 integrates readily within the midplane of the phosphatidylcholine bilayer. These nanodisk structures are defined as "reconstituted high-density lipoproteins" [128], and the CoQ 10 is integrated into a disk-shaped bilayer. The solubilization efficiency of the nanodisk-CoQ 10 was determined to be 97%, as compared to CoQ 10 which is essentially insoluble when dispersed in the presence of just apo-A1 or phosphatidylcholine [126]. The authors then assessed the disk's ability to transport CoQ 10 into HepG2 cells. HepG2 cells exhibited a significant increase in mitochondrial CoQ 10 content upon incubation with nanodisk-associated CoQ 10 compared to empty nanodisks. The authors conclude this nanodisk to be a promising vehicle for exogenous CoQ 10 uptake and distribution in liver epithelium. However, the study lacked a control where CoQ 10 was supplemented in the absence of the nanodisk formation. This would have provided a useful point of comparison.
To further investigate the potential effectiveness of biomolecular nanodisks as a drug-delivery vehicle for CoQ 10 , cultured C2C12 myotubes were treated with suprapharmacological doses of simvastatin, an HMG-CoA reductase inhibitor that decreases the production of polyprenyl diphosphate precursors, and nanodisk-CoQ 10 [127]. Treatment with simvastatin caused a dose-dependent decrease in cell viability, reduced mitochondrial CoQ 10 content, and reduced the oxygen consumption rate. However, co-treatment with simvastatin and nanodisk-CoQ 10 increased mitochondrial CoQ 10 content compared to simvastatin treatment alone and ameliorated the reduction in oxygen consumption rate. These results highlight the potential of nanodisks to dramatically improve the bioavailability of CoQ 10 . It would be interesting to assess the stability of CoQ 10 H 2 in nanodisks and compare its uptake relative to that of the nanodisk-CoQ 10 . Animal studies are needed to determine the efficacy of nanodisks as a drug-delivery vehicle in vivo.

Endothelial Cell Lines
Following absorption of CoQ 10 into the bloodstream, endothelial cells are the next point of uptake. Endothelial cells form the monolayer that line the luminal surface of blood vessels and capillaries, mediating macromolecule transport to and from neighboring tissues. The endothelium is studied extensively in the uptake and distribution of drugs due to this essential role in post-digestive tract drug transport. Human umbilical vein endothelial cells (HUVECs), human corneal endothelial cells, and bovine aortic endothelial cells (BAECs) have been utilized as models for CoQ uptake.
Naguib et al. recently reported the uptake of CoQ 10 H 2 in HCEC-B4G12 human corneal endothelial cells. HCEC-B4G12 cells exhibited a higher increase in total CoQ 10 content when supplemented with CoQ 10 H 2 /γ-cyclodextrin as compared to non-complexed CoQ 10 [120]. Furthermore, HCEC-B4G12 cells treated with ferroptosis-inducer erastin exhibited viability akin to that of untreated cells when incubated with as little as 1 µM CoQ 10 H 2 /γ-cyclodextrin, whereas HCEC-B4G12 cells treated with erastin and 100 µM non-complexed CoQ 10 H 2 exhibited minimal rescue [120]. Uptake was also assessed by measuring ROS production in response to antimycin A, a mitochondrial electron transport chain Complex III inhibitor [120]. HCEC-B4G12 cells demonstrated a dose-dependent rescue of ROS generation in response to supplementation with CoQ 10 H 2 /γ-cyclodextrin, whereas non-complexed CoQ 10 H 2 produced no significant change [120]. The authors report a promising level of CoQ 10 H 2 /γ-cyclodextrin uptake by this corneal endothelial cell line, pointing towards possible exogenous CoQ 10 H 2 formulations involving γ-cyclodextrin to protect against oxidative stress-induced loss of function in human corneas.
BAECs incubated with non-complexed CoQ 10 do not exhibit significant levels of uptake. When BAECs were treated with glucose oxidase, an enzyme that oxidizes glucose to produce hydrogen peroxide, incubation with MitoQ reduced the activity of apoptotic enzymes Caspase-3 and Caspase-9, indicating rescue from oxidative stress [131]. Conversely, incubation with CoQ 10 did not reduce apoptotic activity, suggesting poor uptake of CoQ 10 and/or impaired trafficking to sites essential for protection against oxidative stress [131]. Furthermore, when BAECs pre-treated with 1 µM Mito-Q were incubated in either glucose oxidase or hydroperoxides, complex I activity (as measured via the rate of NADH oxidation) exhibited significant rescue compared to the untreated control; whereas pre-treatment with 1 µM CoQ 10 resulted in insignificant rescue in both glucose oxidase and hydroperoxide incubated BAECs [131]. Perhaps CoQ 10 H 2 /γ-cyclodextrin treatment could improve the uptake in BAECs, like that discussed for CaCo-2 and corneal epithelial cells.

Human Blood Cells
The HL-60 cell line is a human myeloid leukemia cell derived from a patient with promyelocytic leukemia. These cells may be cultured in suspension. The trafficking of endogenously synthesized 14 C-labeled CoQ 10 was compared with the trafficking of exogenously added CoQ 9 [83]. HL-60 cells were incubated with 14 C-labeled 4-HB, the ring precursor of CoQ biosynthesis (Figure 2), and the content of 14 C-labeled CoQ 10 was determined in subcellular fractions over a period of 24 h. Fractions enriched in mitochondria contained more than 90% of the 14 C-labeled CoQ 10 , as ascertained at time points from two to 24 h. Fractions enriched in ER and mitochondria-associated membranes (MAM) contained small amounts of 14 C-labeled CoQ 10 by two hours, and the plasma membrane fraction had detectable 14 C-CoQ 10 only after six hours [83]. HL-60 cells treated with exogenous CoQ 9 showed an accumulation of CoQ 9 in mitochondrial and MAM fractions, followed by its subsequent accumulation in ER and plasma membrane fractions. After twelve hours of incubation with CoQ 9 , the mitochondrial fraction contained the highest content of CoQ 9 , followed by the ER and MAM fractions with similar CoQ 9 content, whereas the plasma membrane fraction exhibited significantly lower CoQ 9 content [83]. The endo-lysosomal membrane fraction was most enriched in CoQ 9 , but this was not tracked during the time course. Treatment of HL-60 cells with brefeldin A, an inhibitor of vesicle formation and transport between the ER and Golgi, prior to the addition of 14 C-4-HB resulted in a profound inhibition in the content of 14 C-CoQ 10 in all fractions, with the plasma membrane fraction being most decreased. It is interesting that inhibition of trafficking between the ER and Golgi had such a dramatic effect on the endogenous synthesis of CoQ 10 within the mitochondria, and may indicate the importance of contact sites between the ER and mitochondria. Treatment of HL-60 cells with brefeldin A and CoQ 9 resulted in most of the CoQ 9 being sequestered in the endo-lysosomal fraction [83]. The high CoQ 9 content in the endo-lysosomal fraction suggests the importance of the endo-lysosomal trafficking pathway in the uptake and assimilation of exogenous CoQ 9 . These findings indicate the importance of a functional endomembrane system in the synthesis of endogenous CoQ 10 and in the transport of both exogenous CoQ 9 and endogenous CoQ 10 .
A recent paper by Wani et al. [132] found exogenous CoQ 10 (non-complexed) to ameliorate oxidative stress in erythrocytes and lymphocytes, suggesting its uptake. Erythrocytes and lymphocytes isolated from donor blood samples were exposed to TiO 2 nanoparticles to induce oxidative stress. Upon treatment with TiO 2 , erythrocytes exhibited impaired ATPase activity and increased ROS production, and lymphocytes had severely depleted ATP levels and reduced mitochondrial membrane potential. Incubation with TiO 2 in the presence of CoQ 10 produced significant rescue of all markers of oxidative stress to wild-type or near-wild-type levels in both erythrocytes and lymphocytes [132]. These data indicate the ability of lymphocytes to take up and distribute exogenous CoQ 10 to the mitochondria, where it restored mitochondrial membrane potential, ATPase activity, and ATP levels. Lymphocytes are also able to traffic exogenous CoQ 10 to other cellular membranes, where it can slow lipid peroxidation induced by oxidative stress.

Human Dermal Cells
Human dermal fibroblasts (HDFs) are specialized cells found in the dermis that play a vital role in the maintenance and repair of the skin. The use of HDFs is especially relevant to investigate the efficacy of CoQ 10 as a commercially available skincare and anti-wrinkle product. HDFs exhibit significant levels of exogenous CoQ 10 uptake, as measured by a reduction in ROS generation [133]. The authors used pollutant particulate matter to activate KU812 cells, a basophilic leukocyte cell line, and used the resulting conditioned medium as a stress treatment of HDFs. Such treatment elicits a proinflammatory response and ROS generation in HDFs. Following eight hours of treatment, the HDFs were then treated with 100 nM CoQ 10 . The exogenous CoQ 10 affected a significant reduction in mitochondrial ROS generation [133]. These findings suggest the ability of HDFs to take up and traffic CoQ 10 to the mitochondria.
HDFs have also been used to demonstrate an efficacious uptake of liposomal CoQ 10 . CoQ 10 was suspended into liposomes via thin-film hydration, with a drug entrapment efficiency of 73.1% [134]. Administration of this liposomal-CoQ 10 suspension to HDFs that were oxidatively stressed from hydrogen peroxide treatment resulted in a complete rescue of cell viability to the point of cell proliferation, whereas non-complexed CoQ 10 administration of the same concentration caused no significant change in cell viability [134]. Similarly, quantification of ROS in these HDFs revealed that liposomal-CoQ 10 supplementation significantly reduced ROS production, while supplementation with non-complexed CoQ 10 produced no significant change [134]. The uptake of CoQ 10 by HDFs is thus augmented by liposomal suspension, where the CoQ 10 can restore antioxidant function.
HDFs exhibit improved uptake of exogenous CoQ 10 H 2 versus CoQ 10 . Incubation of HDFs with simvastatin decreased CoQ 10 levels by 29% [135]. A 24-h supplementation with 15 µg/mL CoQ 10 H 2 caused a dose-dependent increase in total cellular CoQ 10 content, up to an 87-fold increase compared to the vehicle control. In contrast, supplementation with 15 µg/mL CoQ 10 resulted in just a 4.6-fold increase, a 95% reduction compared to CoQ 10 H 2 [135]. Isolated mitochondria in HDFs treated with CoQ 10 H 2 also exhibited a 160-fold increase in total CoQ 10 content, compared to a 2.5-fold increase with CoQ 10 treatment. Thus, treatment with CoQ 10 H 2 resulted in enhanced mitochondrial CoQ 10 content relative to treatment with CoQ 10 [135]. The administration of CoQ 10 H 2 to enhance uptake is promising, but the mechanisms that produce such high uptake relative to CoQ 10 remain uncharacterized.
Keratinocytes are epidermal skin cells that constitute the majority of the human epidermis, essential in forming the primary defense against environmental damage. CoQ 10 treatment has recently been shown to protect keratinocytes from oxidative stress-induced cell death, indicating its uptake [136]. HaCaT human keratinocytes derived from the foreskin were incubated with CoQ 10 directly added to Dulbecco's modified Eagle's Medium (DMEM) and fetal bovine serum (FBS), prior to treatment with the free-radical generating compound 2,2 -azobis-(2-amidinopropane) dihydrochloride (AAPH). Interestingly, no rescue of cell viability was observed until the DMEM-CoQ 10 medium was enhanced with CoQ 10 premixed in FBS, which resulted in a significant rescue of cell viability in the keratinocytes [136]. Similarly, intracellular CoQ 10 H 2 content increased significantly after incubation with the premixed CoQ 10 medium, as compared to both the control and the original, insolubilized CoQ 10 medium [136]. The authors suggest that uptake of CoQ 10 by keratinocytes is augmented by the administration of CoQ 10 in a premixed medium, where otherwise an inefficient uptake is observed.
Keratinocytes have proved useful in the characterization of a new iodine-labeled CoQ derivative that may allow monitoring of the biodistribution of exogenous CoQ [137]. The novel iodine-labeled CoQ 10 (I 2 -Q 10 ) consists of CoQ 10 with two iodine atoms replacing the terminal carbon of the polyisoprenyl tail. Iodine fluoresces upon excitation via X-rays, allowing for quantification and localization of I 2 -Q 10 with minimal overlap between other molecules. Human keratinocytes incubated in 50 µM I 2 -Q 10 showed no signs of toxicity or cell death, with levels of CoQ 10 uptake comparable to that of unlabeled CoQ 10 . X-ray fluorescence imaging of pelleted keratinocytes incubated in I 2 -Q 10 revealed a cell-specific localization of the iodine fluorescence with no extra-pellet iodine leakage. Furthermore, incubation with 50 µM I 2 resulted in a significant decrease in the pellet's fluorescence, demonstrating poor uptake of elemental iodine and thus a mechanism for I 2 -Q 10 uptake that is independent of iodine labeling uptake per se. The authors found exogenous I 2 -Q 10 to distribute homogeneously over the keratinocyte cell, but co-localization with subcellular organelles, such as mitochondria, was not determined. Beyond the confirmation that CoQ 10 is taken up by human keratinocytes, these data suggest a potential way to assess CoQ 10 distribution using I 2 -Q 10 in X-ray fluorescence imaging [137]. The technique was demonstrated to allow for quantitative analysis with no phenotypic changes due to the iodine labeling, and it shows promise in the investigation of mechanisms underlying exogenous CoQ uptake. It is also worth investigating whether the supplementation of with I 2 -Q 10 restores respiratory function in a CoQ-deficient cell culture model, as some CoQ derivatives suffer from a loss of function upon modification of the tail (e.g., Mito-Q).

HeLa Cells
HeLa cells, derived from cervical cancer cells, are the oldest and most generally used human cell line in medicine to date. Recent work in HeLa cells has identified STARD7 as a phosphatidyl transfer protein involved in the export of endogenous CoQ 10 from the mitochondria to the plasma membrane, where it plays a role in ferroptosis suppression. STARD7 is localized to both the mitochondrial intermembrane space and the cytosol (Figure 6a). Deletion of STARD7 causes a nearly 5-fold increase in ferroptosis in response to treatment with PUFAs and erastin [63]. STARD7 is postulated to deliver mitochondrial CoQ 10 to the plasma membrane, where it is reduced by FSP1 to CoQ10 10 H 2 and suppresses ferroptosis. Expression of a cytosol-localized form of STARD7 in HeLa cells significantly decreased cell death in response to treatment with erastin and PUFAs. Inhibition of either CoQ 10 biosynthesis or FSP1 function completely abolished resistance to ferroptosis, resulting in wildtype-levels of cell death in response to oxidative stress [63]. Expression of the mitochondria-localized form of STARD7 alone resulted in a significant decrease in plasma membrane-associated CoQ 10 , while the mitochondrial CoQ 10 content was unaffected. The introduction of cytosol-localized STARD7 rescued plasma membrane-associated CoQ 10 content, suggesting that STARD7 functions as a CoQ 10 transporter in both the mitochondria and cytosol [63].
HeLa cells have also been used to study the effect of supplemented CoQ 10 on sulfide oxidation. SQR is the initial enzyme in the sulfide oxidation pathway, whose impairment can lead to increased ROS. CoQ 10 is an essential cofactor for SQR and protects the cell against oxidative stress. Silencing of COQ8A RNA in HeLa cells decreased the CoQ 10 content by approximately 50% and led to enhanced ROS production. Supplementation of CoQ 10 via Hydro-Q-Sorb decreased ROS production to near-control levels [138]. However, when SQR was knocked down in these CoQ 10 -deficient cells, exogenous CoQ 10 no longer decreased ROS production [138]. This finding indicates that the supplementation of CoQ 10 rescued ROS levels due to CoQ 10 -SQR activity, which requires CoQ 10 localization to the inner mitochondrial membrane. Thus, HeLa cells could take up the Hydro-Q-Sorb CoQ 10 and traffic it to the inner mitochondrial membrane, where it rescued sulfide oxidation.
HeLa cells have been utilized in the characterization of a lipopeptide-based surfactant, caspofungin (CF), that has been shown to solubilize CoQ. CF can form micellized nanoparticles with CoQ 10 up to 200 nm in diameter, with a critical micelle concentration of 50 µM [139]. Wang and Hekimi recently found that CF-solubilized CoQ 10 is a highly efficient formulation for exogenous supplementation in HeLa cells. Upon incubation for one hour, the CoQ 10 content in human HeLa cells increased significantly when treated with CoQ 10 -CF as compared to CoQ 10 , demonstrating the complex's rapid uptake. Recent results further demonstrated that treatment with CoQ 10 -CF restored maximal respiration in CoQ 10 -deficient human fibroblasts derived from a patient harboring a homozygous mutation in the COQ7 gene [140]. The mechanism for CF micellization of CoQ 10 remains unknown, and the potential for CoQ 10 -CF as a therapeutic to treat CoQ 10 deficiencies in patients warrants investigation.

Mouse Embryonic Fibroblasts (MEFs)
Wang et al. found that treatment with CoQ 10 -CF can increase the uptake of exogenous CoQ 10 in MEFs in a dose-dependent manner. Incubation of MEFs for 72 h with 20 µM CF and 0.25 µM CoQ 10 produced a near-doubling in basal and maximal oxygen consumption rate as compared mouse embryonic fibroblasts incubated in just 10 µM CF without exogenous CoQ 10 [139]. After just a one-hour incubation with the CoQ 10 -CF complex, MEF CoQ 10 content was approximately 3-fold higher when compared to MEFs incubated with free CoQ 10 for 48 h, indicating the rapid uptake of the complex.

Receptor-Mediated Uptake of CoQ
Receptor-mediated endocytosis plays a key role in the selective uptake of extracellular molecules, including CoQ 10 . Wainwright et al. [141], used an in vitro blood-brain barrier (BBB) endothelial cell model of CoQ 10 deficiency, and identified lipoprotein-associated uptake and efflux mechanisms regulating CoQ 10 distribution across the BBB (Figure 6b). Porcine brain endothelial cells (PBECs) were grown in monolayer on a permeable membrane (see Figure 5), and transport of CoQ 10 was assessed from both the apical to basal (A to B, blood-to-brain side), and basal to apical (B to A, brain-to-blood side). The transport of non-complexed CoQ 10 across porcine brain endothelial cells (PBEC) was extremely low. Pre-treatment of CoQ 10 with bovine plasma-derived serum led to its association with the VLDL/LDL lipoprotein fractions and increased CoQ 10 transport across the PBEC monolayer by 4-fold, suggesting that CoQ 10 may be transported across the BBB as part of a lipoprotein complex. Transport of serum-treated CoQ 10 in the A to B direction matched transport in the B to A direction, indicating that opposing transport systems produced no net accumulation of CoQ 10 in the brain. This was observed for both the PBEC model and for a mouse BBB cell line, bEnd.3 [141]. Transporters at the BBB generally act together to limit systemic lipoprotein and cholesterol transfer to the brain.
Based on known lipoprotein transporters at the BBB, pharmacological inhibitors were screened for their effect on CoQ 10 transport across bEND.3 cells. BLT-1 is an inhibitor that targets the scavenger receptor (SR-B1), which interacts with HDL on the apical membrane to execute caveolae-mediated lipoprotein influx [142]. RAP was used to inhibit the lowdensity lipoprotein receptor (LDLR) family members, including the LDL receptor-related protein 1 (LRP-1). LRP-1 interacts with a variety of ligands to mediate BBB integrity [143]. FPS-ZM1 was used to inhibit the receptor for advanced glycation end products (RAGE). RAGE mediates the influx and opposes LRP-1-mediated efflux of ApoE and amyloid-beta across the BBB [144]. Upon inhibition of either SR-B1 or RAGE, apical-to-basal transport of CoQ 10 was decreased to 44% and 50% of the control, respectively, whereas basal-to-apical transport exhibited no change [141]. The authors suggested a direction-specific role of both receptors for CoQ 10 transcytosis into the brain. In contrast, inhibition of LRP-1 (and LDLR family members) increased apical-to-basal CoQ 10 transport by 168%, with no significant change in the reverse transport, suggesting that LRP-1 directs CoQ 10 transport out of the brain [141].
Wainwright et al. used pABA, a competitive inhibitor of polyprenyldiphosphate-4hydroxybenzoate transferase (Coq2), to create a CoQ 10 -deficient model of the BBB. Treatment with pABA significantly decreased cellular CoQ 10 content and mitochondrial respiratory chain complex activities in bEND.3 cells [141]. Cells treated with pABA also had reduced tight junction integrity and increased permeability (paracellular transport, Figure 6b), causing a shift in the net transport of CoQ 10 in the blood-to-brain direction. Under these pABA-inhibited conditions, the RAGE receptor was responsible for some of the net CoQ 10 transport, while the SR-B1 receptor-mediated transport was no longer active. However, the use of pABA to inhibit CoQ 10 biosynthesis has possible confounding effects. pABA is a substrate for Coq2, and the resulting 4-amino-3-polyprenylbenzoic acid may exert negative effects on cell endocytic trafficking (see Section 2.1). Studies with mammalian cultured cells show that once pABA is prenylated, it is converted to a redoxactive "dead-end" amino-containing analog of DMQ, which may interfere with respiratory electron transport [47]. Instead of pABA, it has been suggested that 4-nitrobenzoic acid (4-NB) should be used as a competitive inhibitor of Coq2, since it is not prenylated [47].
Interestingly, co-supplementation of CoQ 10 with α-tocopherol or Trolox, a watersoluble analog of α-tocopherol that does not interact with lipoproteins, in pABA-treated and untreated bEND.3 cells caused an increase in CoQ 10 efflux in the brain-to-blood direction. This result suggests that the co-supplementation of CoQ 10 with α-tocopherol for individuals with CoQ 10 deficiency might decrease CoQ 10 delivery to the brain [141]. More work is needed to determine whether this effect is due to excessive antioxidants blocking uptake of lipoproteins containing CoQ 10 or if mechanisms of CoQ 10 uptake are mediated by changes in oxidative stress.
Finally, ABCB1 (P-glycoprotein) was studied since it is reported to decrease CoQ 10 transport across the CaCo-2 intestinal epithelial-barrier model [145]. Inhibition of Pglycoprotein with verapamil had no effect on CoQ 10 transport across the BBB, implying a mechanistic difference between exogenous CoQ 10 uptake in small intestinal epithelial cells versus BBB endothelial cells [141]. However, Wainwright et al. note that in the experiments by Itagaki et al. [145] with CaCo-2, exogenous CoQ 10 was supplemented in its free, un-micellized form, which is not favorable for small intestine uptake CoQ 10 . The existence of brain endothelial membrane receptors that enhance CoQ 10 transcytosis in a selective direction across the BBB opens the possibility for antagonistic and/or agonistic supplements to increase exogenous CoQ 10 delivery to the brain.
CD36 is another receptor that has been identified in the uptake of exogenous CoQ. CD36 is a member of the SR family of transmembrane glycoproteins and a class-B scavenger receptor that exhibits high structural and functional homology to SR-B1 [146]. CD36 transports fatty acids and plays a regulatory role in lipid metabolism [147]. HEK293 human embryonic kidney cells overexpressing CD36 took up 57% more CoQ 9 than wild-type cells [148]. Importantly, receptor-mediated uptake of exogenous CoQ 9 and CoQ 10 was observed only when solubilized with Intralipid ® , an emulsion of soybean oil, glycerol, and egg yolk phospholipids. Receptor-mediated uptake was not reported for solvent-or detergent-solubilized CoQ [148]. Additional work by Anderson et al. [148] reports that CD36 drives CoQ uptake in brown adipose tissue (BAT) and is required for normal BAT function (see Section 4.1).

Saposin B Binds CoQ 10 and Regulates CoQ 10 Content
Prosaposin is a precursor protein that undergoes proteolytic cleavage to generate four small proteins known as saposins (saposins A, B, C, and D). Saposin B is a heatstable glycoprotein that is detected in many tissues. It forms shell-like dimers to bind sphingolipids and glycerol-phospholipids, and primarily localizes to lysosomes [149][150][151]. CoQ 10 bound to saposin B was first identified in protein fractions collected from human urine samples and later detected in cell-lysates from HepG2 hepatocytes and human sperm [65]. CoQ 10 was shown to be transferred to erythrocyte ghost membranes when incubated with saposin B-CoQ 10 complexes at pH 4.5, suggesting that saposin B may play a role in delivery of CoQ 10 to acidic compartments, such as the lysosome [65].
Saposin B has high binding affinity for CoQ 10 as well as CoQ 9 and CoQ 7 isoforms, but a low affinity for α-tocopherol at pH 7.4 [65]. His-tagged saposin B expressed in E. coli was also shown to bind CoQ 10 in in vitro experiments in a pH-dependent manner, and showed that glycosylation of saposin B is not essential for CoQ 10 binding [152]. Saposin B has a comparable affinity for γ-tocopherol and CoQ 10 at neutral and basic conditions, but a higher affinity for γ-tocopherol at acidic conditions, suggesting that the binding affinity of saposin B for γ-tocopherol is greater than that of CoQ 10 [64]. In binding competition assays, two lipids in hexanes were mixed with aqueous saposin B at pH 7.4 and the resulting aqueous saposin B-bound lipids were measured. Interestingly, saposin B bound γ-tocopherol over CoQ 10 despite similar binding affinities and bound α-tocopherol over CoQ 10 despite lower binding affinity [64]. Based on known interactions of saposin B dimers with phospholipids, the authors postulated that saposin B dimers could bind two molecules of CoQ 10 or one molecule of CoQ 10 and two molecules of tocopherol. Their results suggest that the binding of one molecule of CoQ 10 may accelerate the binding of two molecules of α-tocopherol. The crystal structure of saposin B binding chloroquine was later resolved [153] and molecular docking studies identified two binding sites that are accessible to a variety of ligands [154].
Mutations in prosaposin and changes to its cellular content have been reported to affect CoQ 10 levels and mitochondrial function. Knockdown of prosaposin or overexpression of a prosaposin mutant that cannot form saposin B in HepG2 cells causes a significant reduction in both whole-cell and mitochondrial CoQ 10 content [155]. Likewise, overexpression of prosaposin leads to an increase in whole-cell and mitochondrial CoQ 10 content, suggesting that prosaposin and/or saposin B plays a role in regulating CoQ 10 levels [155]. Knockdown of prosaposin in CaCo-2 cells also decreases cellular CoQ 10 content, causing a decrease in ATP formation and glucose consumption [156]. As observed previously, a decrease in ATP formation is associated with disruption in tight junction formation and increased permeability.
More recent work has modeled long-term CoQ 10 deficiency in HepG2 cells and found that decreased CoQ 10 content was associated with decreased prosaposin levels [157]. Treatment with 4-NB decreased CoQ 10 cellular content after three days and decreased prosaposin protein levels after three months. Decreased CoQ 10 content and prosaposin protein levels were observed up to sixteen months with 4-NB treatment. The addition of the CoQ precursor 4-HB to cells treated with 4-NB significantly rescued both CoQ 10 content and prosaposin protein levels. Studies in prosaposin-deficient mice have not investigated CoQ content, though these mice often die one to two days after birth [158]. In summary, the disruption of prosaposin or saposin B decreases CoQ 10 content, indicating that saposin B is a CoQ 10 transporter that plays a role in regulating CoQ 10 levels.

Rodent Models
Mus musculus (mice), Rattus norvegicus (rats), and Cavia porcellus (guinea pigs) are common rodent models for the study of CoQ biosynthesis and uptake. Both mice and rats endogenously synthesize CoQ 9 as the predominant CoQ isoform, with CoQ 10 present as a minor isoform. This is in contrast to humans, guinea pigs, and other mammalian models, where CoQ 10 is the major isoform [8]. Rodent models are also used to study the effect of genetic mutations on endogenous CoQ biosynthesis [159]. Hence, rodent models have been used to assess how CoQ supplements affect the distribution and content of CoQ in tissues in both normal animals as well as those harboring gene mutations causing primary CoQ deficiency [6,160].

Organism-Level Uptake of CoQ
Due to their insolubility in water CoQ 9 and CoQ 10 are poorly bioavailable, necessitating unusually high doses [2,7]. Dosing in humans averages around 200 mg of CoQ 10 taken twice daily. However, when used to treat neurodegenerative diseases, doses can reach up to 3000 mg per day [161]. As a high-molecular-weight, lipophilic molecule, CoQ 10 is poorly absorbed by the gastrointestinal (GI) tract [162]. However, the consumption of CoQ 10 with other dietary lipids, as well as various supplement formulations, can enhance its uptake [7,162].
A majority of rodent studies investigate the effects of CoQ 10 supplements via oral treatment. The addition of CoQ 10 to rodent chow is a common feeding method. Similar to studies in human patients, high doses have been used to study CoQ 10 uptake in mice and rats [162,163]. As the CoQ 10 -supplemented chow is digested, the hydrophobic CoQ 10 is taken up at the GI tract. In male Sprague Dawley rats, CoQ 10 permeability along the GI tract was investigated using a side-by-side diffusion apparatus, in which isolated GI tract tissues were placed in between a CoQ 10 -containing donor medium and a receptor chamber [164]. These studies indicated that the greatest permeability is located at the duodenum, followed by the colon and the ileum.
Within the intestinal enterocytes, absorbed CoQ 10 is likely to be packaged along with other dietary lipids into chylomicrons, which are transported into the lymphatic system [165]. Chylomicrons in the lymph are then delivered to systemic circulation via the thoracic duct and subjected to processing by lipoprotein lipase in the capillaries. The resulting chylomicron remnants are taken up by the liver (Figure 7) [166]. This delivery may be responsible for CoQ 10 enrichment seen in the liver. We note here the assumption that orally supplemented CoQ 10 is trafficked in a manner similar to other dietary lipids such as vitamin E [167] and cholesterol [168]. However, there is a lack of direct experimental support for the presence of supplemented CoQ 10 in chylomicron remnants. It is important to note that many studies in mice and rats utilize CoQ10 as the supplemented isoform, although the predominant endogenous isoform is CoQ9. This may generate conflicting outcomes when comparing rats or mice with guinea pigs, which solely produce CoQ10 as the endogenous isoform. Several studies in both rats and mice have shown that supplementation of CoQ10 increases endogenous CoQ9 levels in various tissues, serum, and mitochondria [163,173,174]. This is likely due to a sparing effect of supplemented CoQ10 on the endogenous CoQ9 pool in which the addition of CoQ10 to the cellular CoQ pool "spares" the turnover of CoQ9; a similar phenomenon is observed when CoQ10 supplements preserve levels of α-tocopherol or the tissue content of CoQ9 [175,176]. The cardioprotective effects of CoQ9 and CoQ10 supplementation were investigated in guinea pigs [177]. CoQ9 and CoQ10 were found to have functionally identical effects, as both isoforms conferred comparable cardioprotection "as evidenced from the comparable degree of the postischemic ventricular recovery and reduction in myocardial infarct size In the liver, CoQ 10 derived from dietary sources is likely to be combined with de novo synthesized CoQ where it would be assembled into lipoprotein particles, with the majority in very low-density lipoprotein (VLDL) [164]. VLDL secreted by the liver is processed by lipoprotein lipase in the capillaries of various tissues to form intermediate-density lipoprotein and low-density lipoprotein (LDL) [168]. Lipoprotein particles extracted from human plasma find LDL to be most enriched in CoQ 10 content [169]. CoQ 10 H 2 is an important antioxidant in circulating lipoproteins and enhances the resistance of LDL to lipid oxidation [170,171].
High-density lipoprotein particles (HDL) isolated from human plasma contain approximately 50% of the CoQ 10 content as compared to LDL [169]. This relationship between LDL and HDL CoQ content is also observed in mice [148]. In a mouse model, the absence of apolipoprotein ApoA1, a major component of HDL, decreased mitochondrial cardiac CoQ content by 75% [172]. Following ischemia/reperfusion of the left ascending coronary artery, the infarct size increased significantly in the homozygous ApoA1 −/− mice, as compared to the heterozygous ApoA1 −/+ mice, which in turn had larger infarcts than the parental control (WT) mice [172]. Although oral supplementation of CoQ 10 in ApoA1 −/− mice did not affect the cardiac CoQ 10 content, intraperitoneal (IP) injection of CoQ 10 restored the cardiac mitochondrial CoQ 10 content of the ApoA1 −/− mice to WT levels, and decreased their infarct size to that of WT mice following ischemia-reperfusion [172]. These results suggest a possible role for ApoA1 in the uptake and trafficking of CoQ 10 from the GI tract to the heart; however, further study is needed to confirm this relationship. It is unfortunate that in this study the authors did not quantify the endogenous CoQ 9 content as it may have further informed the outcome of supplementation with CoQ 10 . However, another confounding factor is that the authors used CoQ 9 as an internal standard for the quantification of CoQ 10 . This is problematic for two reasons: (1) mouse heart contains endogenous CoQ 9 , and (2) commercially available standards of CoQ 9 can be contaminated with CoQ 10 .
It is important to note that many studies in mice and rats utilize CoQ 10 as the supplemented isoform, although the predominant endogenous isoform is CoQ 9 . This may generate conflicting outcomes when comparing rats or mice with guinea pigs, which solely produce CoQ 10 as the endogenous isoform. Several studies in both rats and mice have shown that supplementation of CoQ 10 increases endogenous CoQ 9 levels in various tissues, serum, and mitochondria [163,173,174]. This is likely due to a sparing effect of supplemented CoQ 10 on the endogenous CoQ 9 pool in which the addition of CoQ 10 to the cellular CoQ pool "spares" the turnover of CoQ 9 ; a similar phenomenon is observed when CoQ 10 supplements preserve levels of α-tocopherol or the tissue content of CoQ 9 [175,176]. The cardioprotective effects of CoQ 9 and CoQ 10 supplementation were investigated in guinea pigs [177]. CoQ 9 and CoQ 10 were found to have functionally identical effects, as both isoforms conferred comparable cardioprotection "as evidenced from the comparable degree of the postischemic ventricular recovery and reduction in myocardial infarct size and cardiomyocyte apoptosis". Lekli et al. further claimed that CoQ 9 can be converted to CoQ 10 after supplementation, although no metabolic labeling of the fed CoQ 9 was performed to enable a more substantive conclusion. It is more likely the elevation in CoQ 10 content was due to the aforementioned sparing effect.
CoQ 10 present in lipoproteins may be delivered to cells via receptor-mediated uptake, to facilitate the entry of CoQ at the cellular level [2]. A specific example is the CD36 scavenger receptor-mediated uptake of exogenously supplied CoQ 10 into mouse brown adipose tissue (BAT) [148]. CD36 is present in many tissues, with the highest abundance in adipose tissue, heart, lung, and muscle, and less expression in spleen and liver [148]. BAT functions in non-shivering thermogenesis, a process generated by uncoupled respiration [178]. To perform this function, BAT depends on the uptake of CoQ rather than through its own de novo biosynthesis [148]. BAT isolated from mice lacking the CD36 receptor (CD36 −/− ), have significantly reduced endogenous CoQ levels. Uptake of Intralipid ® -solubilized CoQ 10 in CD36 −/− mice via intraperitoneal injection is significantly reduced in BAT compared to wild-type mice, while no difference was observed in liver or muscle tissues. Moreover, CD36 −/− mice show a significant increase in CoQ 10 serum levels after intraperitoneal injection of exogenous CoQ 10 when compared to wild-type mice, suggesting that BAT is a major sink for exogenous CoQ. CD36 −/− BAT mitochondria showed impaired substrate utilization and decreased basal rates of respiration, which was rescued upon the addition of CoQ 2 . CD36 is found to be exclusively on the plasma membrane in BAT, and BAT lacking CD36 lacks the characteristic browning of BAT following cold exposure. Likewise, CD36 −/− mice exhibit defective non-shivering thermogenesis. The mechanism by which CD36 mediates the uptake of CoQ 9 or CoQ 10 into BAT is not clear. The authors suggested that uptake could proceed either via CD36-mediated endocytosis of lipoprotein particles or that CoQ could be selectively taken up from a bound lipoprotein [148]. In either case, these findings provide evidence for receptor-mediated uptake of CoQ. (See Section 3.2.1 for further discussion of receptor-mediated uptake of CoQ 10 at the blood-brain barrier.) Extensive reviews and literature compare the efficacy of supplementation with CoQ 10 and CoQ 10 H 2 supplementation in humans [2,7,179]. However, few studies investigate the relative bioavailability of CoQ 10 H 2 relative to CoQ 10 in a rodent model. In rats, CoQ 10 is converted into CoQ 10 H 2 during or after absorption by the intestine [162], yet exogenous supplementation with CoQ 10 H 2 has been posited to increase the molecule's bioavailability. Mice lacking the ability to synthesize CoQ due to homozygous partial loss of function mutations in Coq9 (Coq9 −/− ) were supplemented with water-soluble formulations of both CoQ 10 and CoQ 10 H 2 [180]. While both formulations increased tissue levels relative to the Coq9 −/− control, the tissues of mice fed CoQ 10 H 2 tended to have greater enrichment in CoQ 10 compared to mice fed CoQ 10 [180]. Measurement of cerebrum mitochondrial CoQ 10 content revealed the same trends. A recent study used positron emission tomography imaging to detect the uptake and distribution of 11 C-CoQ 10 and 11 C-CoQ 10 H 2 that were administered to rats via tail vein injection [181]. The uptake of the labeled CoQ 10 H 2 in the cerebrum, cerebellum, white adipose tissue, muscle, kidney, testis, and BAT was higher than that of the labeled CoQ 10 . However, uptake of the 11 C-labeled CoQ 10 H 2 was lower in the spleen as compared to labeled CoQ 10 . Although the delivery of CoQ 10 H 2 in this 11 C-labeling study was via intravenous injection [181], it is interesting that the enrichment achieved by CoQ 10 H 2 in brain tissues was also observed for diet-delivered CoQ 10 H 2 in the Coq9 −/− mouse study [180].

Tissue Localization of Supplemented CoQ
Understanding the localization of supplemented CoQ to specific tissues may be important to treating primary CoQ deficiencies that affect specific or multiple organs. However, as discussed above, the enrichment of CoQ in specific tissues of rats and mice can be affected by the redox state of the supplement. There is also variability observed in rats, mice, and guinea pigs following supplementation with different formulations of CoQ. This variability can be attributed to the conditions of supplementation including dose, mode of supplement administration, lipid extraction, and quantification methods used, as well as the genetic background or strain of the animal model.

Rat
IP injection into rats of 3 H-CoQ 10 , labeled at the isoprenyl methylene closest to the benzoquinone ring, permitted quick evaluation and extensive uptake into a number of tissues. Enriched tissues include the liver, spleen, adrenals, and ovaries, with limited uptake into the heart and thymus, as well as white blood cells [182]. This is in contrast to the poor dietary uptake of 3 H-CoQ 10 . Over a two-week period of oral supplementation, only 4-6% was taken up while the rest was excreted as radioactive metabolites in urine and feces. Analysis of the excreta revealed that half of the radioactive content excreted was present as non-metabolized CoQ 10 in feces, suggesting that excess CoQ 10 is removed through bile excretion (Figure 7) [182]. The results concur with prior studies that showed both oral and IP injections of CoQ 10 resulted in liver and spleen enrichment, while none was detected in the kidney, heart, or brain tissue [173,183]. However, Kwong et al. [174] observed modest CoQ 10 enrichment of the heart and kidney tissues. Differences in enrichment between studies may be attributed to dosage and method of feeding. Reahal [174]. CoQ 10 is better taken up as an emulsion compared to a powder [2,7].
Intravenous (IV) administration in rats of 4 mg/kg CoQ 10 solubilized in lipid microspheres or chromophore EL has also been investigated [184]. Chromophore EL was described as a polyethoxylated castor oil that contained approximately 40 units of ethylene oxide to each triglyceride unit along with the addition of undefined antioxidants [185]. Results from Scalori et al. [184] indicate that CoQ 10 , whether orally or IV administered, leaves the blood plasma quickly and is particularly enriched in the liver, although with varying kinetic profiles. The heart and kidneys of these rats were also enriched in CoQ 10 . However, smaller doses of CoQ 10 were needed to achieve the same effect when supplemented by IV [184].
Topical application of CoQ 10 , a lesser-studied method of administration, has also been found to increase skin concentrations in Sprague Dawley rats [184]. This is consistent with more recent data indicating that topical administration of CoQ 10 on the forearms of human females significantly increased levels at the skin surface and the deeper epidermis layer [186], and reduced UVB-induced wrinkle formation [187].

Mouse
Similar results for tissue enrichment of CoQ are seen in mouse models. Following a 13-week regimen of CoQ 10 supplementation in a soybean oil suspension, the serum and liver homogenate had increased levels of CoQ 10 [175]. No changes to CoQ 10 content were observed in the brain, heart, kidney, or skeletal muscle homogenates. However, a higher dose, 200 mg/kg/day, and a longer regimen of CoQ 10 were shown to increase cerebral cortex levels of CoQ 10 , CoQ 10 H 2 , and CoQ 9 H 2 [188]. These results are recapitulated in a study looking at the short-term administration of CoQ 10 H 2 [189]. Imaging mass spectrometry also shows increased CoQ 10 H 2 in several regions of the brain, including the hippocampus, cerebellum, and brain stem [189]. Matthews et al. showed oral supplementation of CoQ 10 exerted neuroprotective effects by reducing striated lesion volumes produced from systematic administration of 3-nitropropionic acid, a complex II inhibitor [188].

Guinea Pig
In the guinea pig model, Passi et al. [190] investigated the effect of CoQ 10 and ubiquinol 10 -diacetate (CoQ 10 -DIA) provided in the diet, on liver subcellular fraction content. CoQ 10 H 2 modified by acetate at the ring 1,4 positions, (CoQ 10 -DIA) is readily hydrolyzed and taken up as CoQ 10 H 2 . The study found that both CoQ 10 and CoQ 10 -DIA taken up were mainly enriched in the heavy mitochondria and light mitochondria/lysosome fractions. Modest increases in CoQ 10 content were also seen in the nucleus/debris and microsome fractions. There were no significant differences in subcellular enrichment between CoQ 10 and CoQ 10 -DIA [190].
Across all three rodent models, exogenously supplemented CoQ 10 is enriched mainly in the liver, plasma, and serum. While there remains conflicting data on CoQ enrichment in other tissues, it appears that high dosage, IV, or IP administration may be able to overcome certain barriers to uptake into tissues such as the brain, heart, and kidney.

Subcellular Localization of Supplemented CoQ
When the CoQ sequestered in lipoproteins is taken up by a cell, it must be trafficked to intracellular membranes. Currently, little is known about the mechanisms of intracellular trafficking used to shuttle CoQ. However, the subcellular enrichment of CoQ following supplementation has been studied in mice, rats, and guinea pigs.

Rat
Under normal physiological conditions, endogenous CoQ 9 in rat liver tissue is predominantly located in the mitochondrial subcellular fraction [183]. After CoQ 10 supplementation, subcellular fractionation of hepatic tissue cells was investigated [191]. The pellet containing lysosomes and light mitochondria was especially enriched in CoQ 10 , with no change to the endogenous levels of CoQ 9 . Bentinger et al. [182] conducted an investigation into the subcellular localization of 3 H-CoQ 10 in the liver, finding that nearly 60% of radiolabeled CoQ 10 localized to the lysosome after IP supplementation, whereas the mitochondrial fractions contained only approximately 11% of the total 3 H-CoQ 10 recovered [182]. This is consistent with a prior study, where IP administered CoQ 10 was primarily localized to the soluble cytosolic fraction of liver cells and was recovered as the oxidized form, not the reduced form [183]. As CoQ 10 is highly hydrophobic, it is unclear why the concentration was found to be greatest in the soluble cytosolic fraction. It is also worth mentioning that this study does not indicate precautions taken to mitigate autoxidation of reduced CoQ 10 H 2 in liver extracts during tissue homogenization and lipid extraction. Such precautions may include quick procedural timing, the use of acidified solvents during extraction, and inert gases to prevent autoxidation. An LC-MS/MS quantification method sensitive to the CoQ redox state was recently developed to minimize measurement error due to autoxidation [119].
In a separate rat study, the brain and brain mitochondria were shown to be enriched in CoQ 10 following a two-month supplementation regimen at a dose of 200 mg CoQ 10 /kg per day [188]. However, the CoQ 9 content was not significantly increased in this study. Another study administering 150 mg/kg CoQ 10 per day for 13 weeks demonstrated an increase in heart, skeletal muscle, liver, and kidney mitochondrial CoQ 10 content, as well as an additional increase in CoQ 9 content in mitochondria of heart, skeletal muscle, and kidney tissue homogenate [174]. Only the mitochondria-enriched fractions were examined for CoQ 9 and CoQ 10 contents in these two studies.

Mouse
Mice supplemented with CoQ 10 generally presented with increases in both CoQ 9 and CoQ 10 content in the liver, heart, kidney, and skeletal muscle tissue mitochondria, but not in the brain [163,176]. The data obtained by Sohal et al. were subjected to further analyses to consider the length of treatment and dosage factors. General trends indicated that mice treated for 17.5 months with CoQ 10 had larger magnitude increases in CoQ content as compared to mice treated for 3.5 months [163]. No consistent dose-dependent trend was reported. However, these conclusions are solely based on the examination of mitochondria-enriched fractions

Guinea Pig
In the guinea pig model, Passi et al. [190] investigated the effect of dietary CoQ 10 and CoQ 10 -DIA on liver subcellular fraction content. The authors found that both CoQ 10 and CoQ 10 -DIA taken up were mainly enriched in the heavy mitochondria and light mitochondria/lysosome fractions. Modest increases in CoQ 10 content were also seen in the nucleus/debris and microsome fractions. There were no significant differences in subcellular enrichment between CoQ 10 and CoQ 10 -DIA.

Intracellular Trafficking of CoQ
There is little information on the intracellular trafficking of exogenously supplemented or endogenously synthesized CoQ in rodent models. Data gleaned from other cellular models, including Saccharomyces cerevisiae and human cell lines, can inform such studies. Intriguingly, ADCK2, the mouse homolog of yeast Cqd1 (discussed in Section 2.3), has been shown to play a role in modulating the transport of lipids into mitochondria for fatty acid β-oxidation and CoQ biosynthesis [95]. ADCK2 and Cqd1 are members of the UbiB/Coq8 family of atypical kinases. Adck2 +/− mice exhibit haploinsufficiency, with CoQ deficiency specifically in skeletal muscle and defects in mitochondrial fatty acid oxidation [95]. CoQ 10 supplementation increased the content of CoQ 10 in the skeletal muscle of the Adck2 +/− mice, enhanced physical performance, and decreased plasma lactate levels [95]. It seems likely that ADCK2 acts similarly to Cqd1, the yeast ortholog, and is necessary to retain CoQ within mitochondria [93].

Mouse Models of CoQ Deficiency and the Effects of CoQ Supplementation
Investigation of human mutations in COQ genes has been studied in transgenic mouse models to assess the organism-level effect of canonical single nucleotide variants. Transgenic mice with homologous gene KOs have also been used to assess and investigate phenotypic consequences, and to evaluate the efficacy of treatment with CoQ 10 supplements.
Saiki et al. [192] investigated the efficacy of CoQ 10 supplementation in the Pdss2 kd/kd (kidney disease) mouse. The Pdss2 kd/kd mouse harbors a spontaneous homozygous V117M mutation resulting in a partial loss of function in PDSS2, one of the two subunits of polyprenyldiphosphate synthase essential for CoQ biosynthesis, and a homolog of yeast Coq1 [192,193]. Pdss2 kd/kd mice present with CoQ deficiency in early adulthood, resulting in a kidney disease similar to the collapsing glomerulopathy variant of focal segmental glomerulosclerosis [192]. Mice harboring a podocyte-specific loss of function knockout of Pdss2 reproduced a more severe phenotype of the Pdss2 kd/kd mice, indicating that the kidney disease in the Pdss2 kd/kd mouse results from the CoQ deficiency in the podocytes [193]. Intriguingly, supplementation with CoQ 10 afforded a dramatic rescue of the kidney disease as ascertained by a 5-fold reduction in urine albumin content. However, CoQ 10 supplementation, even at doses of 200 mg/kg per day did not affect the kidney CoQ 10 content as compared to the untreated Pdss2 kd/kd mouse. It was speculated that the CoQ 10 supplementation may exert its effects via the filtration of blood plasma components [194]. The podocyte foot processes act as a filtration barrier, and the CoQ 10 may function primarily as a membrane antioxidant [192]. A more recent study using the same Pdss2 kd/kd mouse model supports CoQ 10 supplementation (0.5% CoQ 10 supplemented food), as a preventative treatment for the progression of nephrotic syndrome induced by deficiency [138]. Kidney CoQ 9 and CoQ 10 levels were increased, and damage indicated by renal histology is significantly dampened after six months of supplementation with CoQ 10 . CoQ 10 supplementation rescued defects in the oxidation of sulfide, and decreased oxidative stress in the kidneys of Pdss2 kd/kd mice [138].
The kidneys of Pdss2 kd/kd mice are shown to have a defect in the sulfide oxidation pathway resulting in increased hydrogen sulfide and depleted glutathione [195]. Deficiencies in CoQ impede the sulfide oxidation pathway by decreasing SQR activity [195,196]. Further investigation of mouse Coq9 mutants revealed similar results. Coq9 R239X mice, with 10-15% residual CoQ 9 content, had significantly less SQR levels in the kidneys and cerebrum whereas Coq9 Q95X , with 40-50% residual CoQ 9 content in the same tissues, had no decrease in these tissues [196]. Both models exhibit 10-20% residual CoQ 9 content in muscle and have decreased SQR in this tissue. While low CoQ content disrupts sulfide oxidation, supplementation with 100 µM CoQ 10 upregulates the sulfide oxidation pathway, restoring its function as well as other downstream and tangential effects of impaired sulfur metabolism [197].
Investigation of the mouse Coq7 homolog, Mclk1, showed that homozygous knockout resulted in embryonic lethality [198]. Heterozygous Mclk1 +/− mice, however, live a normal lifespan with no significant defects in subcellular CoQ 9 levels or mitochondrial morphology. Subcellular distribution of CoQ 9 is altered, which may explain the lowered oxygen consumption and ATP production phenotype of heterozygous Mclk1 +/− mitochondria [199,200]. Supplementation with CoQ 10 significantly increases mitochondrial CoQ 10 content and restores the electron transport chain defect [198].
Rare deleterious mutations in the human COQ9 gene result in severe multisystem disorders, affecting the central nervous system, cardiac, and renal systems [201]. Homozygous Coq9 mice (Coq9 R239X/R239X ) were prepared to model the human COQ9 R244X deficiency [180]. These mice exhibit profound brain damage due to oxidative damage, neuronal apoptosis, and demyelination in the pons and medulla oblongata [180]. After two months of 240 mg/kg/day CoQ 10 or CoQ 10 H 2 oral supplementation, significant increases were detected in plasma, liver, and muscle tissue, followed by smaller increases in the heart, kidney, cerebrum, and cerebellum [180]. Supplementation with CoQ 10 H 2 appeared to alleviate histopathological symptoms of CoQ deficiency in the brain, whereas CoQ 10 did so to a lesser extent.
Such dramatic effects of CoQ 10 supplementation have also been seen in some human patients harboring primary CoQ deficiency [202][203][204][205]. The parallels between the rodent disease models and the human clinical outcomes emphasize the benefit of CoQ-deficient mouse research models.

Conclusions
CoQ is an essential and ubiquitous lipid with many complex roles. However, there remains much to be learned about the mechanisms responsible for its uptake, intracellular trafficking, and distribution. Here, we have summarized the work in several model organisms used to investigate how CoQ is trafficked.
Yeast is an extraordinarily powerful model organism that has been used to identify proteins involved in the uptake and trafficking of exogenous CoQ 6 to mitochondrial respiratory complexes. The yeast coq mutants completely lack CoQ 6 biosynthesis and are thus respiratory deficient and fail to grow in a medium containing a non-fermentable carbon source. Supplementation with exogenous CoQ restores respiration and growth of coq mutants, and supplementation with the soluble CoQ 2 analog can bypass defects in endocytosis and membrane trafficking [18,19,58]. Fernandez-del-Rio et al. [18] identified six genes that are essential for the uptake and trafficking of exogenous CoQ 6 to mitochondrial respiratory complexes-RTS1, CDC10, RVS161, RVS167, VPS1, and NAT3. More work is needed to determine the role that each of these genes plays in the uptake and trafficking of exogenous CoQ 6 . While supplementation of ORF∆coq2∆ double mutants with exogenous CoQ 6 is a powerful approach for identifying genes required for CoQ 6 uptake and trafficking, genes that are redundant or essential for respiration cannot be identified with exogenous CoQ 6 rescue screens.
Yeast is also a powerful model for determining pathways and proteins responsible for the trafficking of endogenously synthesized CoQ 6 . Biosynthesis and transport of endogenously synthesized CoQ 6 from the mitochondria in yeast is possibly mediated by ERMES [88,89]. However, direct transport of CoQ 6 through ERMES has not yet been observed. Cqd1 and Cqd2 are mitochondrial inner membrane proteins that face the intermembrane space and influence the cellular distribution of endogenous CoQ 6 , and likely play a role in transporting CoQ 6 from the mitochondrial inner membrane to the outer membrane and ER [93]. It is likely that other still unidentified CoQ 6 transporters may contribute to trafficking.
Mammalian cell lines are an ideal model for investigating the cellular uptake and intracellular distribution of CoQ. Synthetic vehicles, such as cyclodextrin, caspofungin, micelles, liposomes, and nanodisks have been shown to dramatically improve the bioavailability of exogenous CoQ [117]. Moreover, supplementation with CoQ 10 H 2 displays improved bioavailability in human dermal fibroblasts compared to CoQ 10 [135], a trend that is also observed in rodent models [180,181]. Treatment of HL-60 cells with brefeldin-A shows that intracellular distribution of both endogenous and exogenous CoQ relies at least in part on the endomembrane system [83], though more work is needed to identify essential players of this pathway for CoQ transport. Recent work in HeLa cells has identified STARD7 as a phosphatidyl transfer protein involved in the export of endogenous CoQ from the mitochondria to the plasma membrane, where it plays a role in ferroptosis suppression [63]. Work in mouse endothelial brain cells has identified scavenger receptor SR-B1, receptor for advanced glycation endproducts (RAGE), and LRP-1 to be involved in transport of lipoprotein-associated CoQ 10 across the blood-brain barrier [141]. CoQ 10 may also be delivered to BAT cells by receptor-mediated uptake, via-the CD36 scavenger receptor [2,148].
Rodent models are useful for investigating the bioavailability of CoQ supplements and CoQ distribution in tissues of both wild-type animals as well as those harboring gene mutations that cause primary CoQ 10 deficiency [6,160]. Dietary CoQ in rodents is taken up at the GI tract [164], and is then likely packaged into chylomicrons by intestinal enterocytes and transported into the lymphatic system, though more work needs to be performed to identify the pathways of endothelial uptake and to detect the supplemented CoQ in chylomicrons. CoQ from chylomicron remnants is likely repackaged with de novo CoQ at the liver and secreted in lipoprotein particles, with the majority in VLDL. Tissue localization of supplemented CoQ in rodents varies across studies and CoQ formulations, but enrichment is usually observed in the liver, plasma, and serum, with varying results in other tissues such as the heart, brain, and kidney. Supplemented CoQ is enriched in subcellular mitochondrial and light mitochondrial/lysosomal fractions. Moreover, transgenic mouse models have been used to investigate human mutations and canonical single nucleotide variants in Coq genes in order to evaluate the efficacy of treatment of CoQ-deficient mouse models with CoQ 10 supplements.
It is important to note that comparisons across studies are challenging due to variability in CoQ formulations and dosage. Comparative studies of CoQ 10 and CoQ 10 H 2 must use proper precautions and controls to prevent rapid oxidation of CoQ 10 H 2 . Unfortunately, rodent studies that investigate CoQ 10 uptake sometimes overlook endogenous CoQ 9 content, despite CoQ 9 being the predominant isoform in mice and rats. Many aspects of CoQ uptake and transport remain unknown or poorly understood. What is the relative importance of the endomembrane system and membrane contact sites in the transport of CoQ? How are CoQ transport and intracellular distribution of CoQ regulated? Elucidating the pathways of how organisms assimilate CoQ supplements, its cellular uptake, and pathways of intracellular distribution is important to understanding the functional roles of CoQ and may lead to improved therapeutics for exogenous supplementation.

Acknowledgments:
We thank Greg Payne, Lucia Fernandez-del-Rio, Noelle Alexa Novales, Sining (Cindy) Wang, Kelsey Feustel, and other members of the C.F. Clarke lab for helpful advice and feedback on drafts of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.