**2. CL Biosynthesis**

Mitochondrial membranes are characterized by their high content of membrane proteins and unique phospholipid composition. A recent study found the respiratory chain, the ADP/ATP carrier, phosphatidylethanolamine, phosphatidylcholine and cardiolipin to be the main constituents of the inner membrane [10]. The hallmark lipid is the dimeric phospholipid cardiolipin (CL), which is found almost exclusively in the inner mitochondrial membrane. Four different fatty acids can be bound to CL forming different CL species, leading to a highly diversified CL pool [11]. CL in the colon has short and mostly saturated fatty acids, whereas CL in the brain primarily constitutes long and unsaturated CL species [12]. The heart has an unique CL pool consisting mostly of tetralinoleoyl-CL (CL(18:2)). Interestingly, the highest proportions of oxidized CL were found in heart (1.8% ± 0.7%) and skeletal muscle [12]. The diversified CL pool is acquired by reshaping CL acyl composition after its initial biosynthesis. CL is synthesized by enzymes located in the inner mitochondrial membrane. An important step in the biosynthesis of CL in the inner membrane is catalyzed by PTPMT1, converting phosphatidylglycerol phosphate to phosphatidylglycerol (Figure 1). Deletion of this in mice is embryonically lethal and PTPMT1-deficient Mouse Embryonal Fibroblasts (MEFs) of embryos obtained from intercrosses of PTPMT1+/flox mice reveal the essential role of this enzyme in CL biosynthesis and affect mitochondrial morphology and respiration [13]. CL Synthase (CLS1) catalyzes the subsequent addition of a second molecule of CDP-DAG to form premature CL (Figure 1). The embryonic lethality of CLS1-deficient animals underscores the essential role of CLS1 in CL biosynthesis. Neuron-specific knockout results in neuronal loss and gliosis in the forebrains as a result of defective respiration and morphological abnormalities in these mitochondria [14].

– **Figure 1.** Biosynthesis and remodeling of cardiolipin (CL) in the inner membrane. Phosphatic acid (PA) as a precursor reaches the mitochondrial matrix via the PRELID–TRIAP1 complex. The CDP-DAG synthase TAMM41 activates PA with cytidine triphosphate (CTP), producing CDP-DAG. Subsequently, the phosphatidylglycerol phosphate synthase 1 (PGS1) catalyzes the formation of phosphatidylglycerol phosphate (PGP), followed by the dephosphorylation generating phosphatidylglycerol (PG) via the mitochondrial protein-tyrosine phosphatase 1 (PTPMT1). In a last reaction, Cardiolipin synthase 1 (CLS1) mediates the emergence of premature CL based on one molecule of PG and one CDG-DAG. Following the initial synthesis, CL remodeling ensues. After the removement of fatty acids (FAs) mediated by a member of the phospholipase iPLA2 family, the incorporation of new FAs is proceeded by Tafazzin to form matured CL from Monolysocardiolipn (MLCL). To reach an energetic optimum, the remodeling and the ongoing exchange of FAs is a continual persisting process, culminating in a highly diversified CL pool.

– Afterwards, CL remodeling is initiated by deacylation, catalyzed by mitochondrial members of the Ca <sup>2</sup><sup>+</sup> independent phospholipases to form monolysocardiolipin (MLCL). Subsequently, CL is reacetylated by Tafazzin to form mature CL (Figure 1).

's – The protein Tafazzin is produced by alternative splicing of the TAZ gene. The gene consists of 11 exons and two ATG initiation sites. Transcription of the TAZ gene gives rise to multiple mRNAs by alternative splicing at exons 5–7. A highly hydrophobic segment of 30 residues at the *N*-terminus acts as a membrane anchor. Tafazzin is directed to mitochondria with the help of two independent targeting sequences [15]. The active site of phospholipid-binding has been identified in a 57-amino acid cleft containing positively charged residues [16]. The substrate specificity of Tafazzin was found to be surprisingly low and cannot explain the tissue-specific remodeling of the CL pool [17]. Recent data suggest that Tafazzin's substrate specificity is determined by the thermodynamic properties of lipid domains in the vicinity of the enzyme [17]. Tafazzin exchanges fatty acids in the CL molecule until the molecular species composition with optimal packing conditions and the lowest free energy is established [18]. A recent study from the Schlame lab suggests that the thermodynamic properties of lipid domains are determined by the assembly of respiratory chain complexes. This study proposes that protein crowding in the respiratory chain imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid–protein complexes [19]. Further evidence was provided by an integrative approach of lipidomics and transcriptomics. Oemer et al. found that a transcriptional regulation of CL biosynthesis genes shows no correlation with the tissue-specific CL composition. Interestingly, the only significant compliance with CL content was found for genes of the respiratory chain. This finding supports the idea that the respiratory chain and other protein complexes determine the tissue-specific CL composition of the membrane and hence its properties.

Besides Tafazzin, two other enzymes are involved in CL remodeling. The Coenzyme A-dependent monolysocardiolipin acyltransferase (MLCLAT1) is a splice variant of the HADHA gene encoding for the α-subunit of the human trifunctional enzyme (α-TFP) [20,21]. In a recent study, Miklas et al. show that apart from its function in the β-oxidation cycle, α-TFP plays a role in CL remodeling [22]. As patient-derived HADHA knockout cardiomyocytes do not accumulate MLCL as TAZ mutant cells, it has been concluded that HADHA acts subsequently to TAZ to mature the CL pool. The second enzyme is the ER-MAM resident lysocardiolipin acyltransferase (ALCAT1). Overexpression of ALCAT1 in mouse myoblasts increases the incorporation of docosahexaenoic acid (22:6) into CL, yielding a peroxidation prone form of CL, which has been suggested to reflect a pathogenic CL remodeling [23].
