Cardiolipin-dependent formation of mitochondrial respiratory supercomplexes

Abstract

The organization of individual respiratory Complexes I, III, and IV (mammalian cells) or III and IV (yeast) of the mitochondria into higher order supercomplexes (SCs) is generally accepted. However, the factors that regulate SC formation and the functional significance of SCs are not well understood. The mitochondrial signature phospholipid cardiolipin (CL) plays a central role in formation and stability of respiratory SCs from yeast to man. Studies in yeast mutants in which the CL level can be regulated displayed a direct correlation between CL levels and SC formation. Disease states in which CL levels are reduced also show defects in SC formation. Three-dimensional density maps of yeast and bovine SCs by electron cryo-microscopy show gaps between the transmembrane-localized interfaces of individual complexes consistent with the large excess of CL in SCs over that integrated into the structure of individual respiratory complexes. Finally, the yeast SC composed of Complex III and two Complexes IV was reconstituted in liposomes from purified individual complexes containing integrated CLs. Reconstitution was wholly dependent on inclusion of additional CL in the liposomes. Therefore, non-integral CL molecules play an important role in SC formation and may be involved in regulation of SC stability under metabolic conditions where CL levels fluctuate.

Keywords: Cardiolipin; In vitro reconstitution; Mitochondria; Respiratory supercomplex; Structural analysis.

Figure 1

Biosynthesis and structure of CL. The pathway, genes (red) and gene products responsible for CL synthesis in yeast mitochondria are shown (Henry et al., 2012; Tamura et al., 2013). The pathway in higher eukaryotes is essentially the same and is carried out by homologous genes and gene products. The CLD1 gene product (CL specific deacylase) initiates the remodeling of CL fatty acid chain composition in yeast by forming monolyso-CL (Beranek et al., 2009); higher eukaryotes utilize multiple deacylases (Baile et al., 2013). The TAZ gene product in both yeast and mammalian cells is responsible for completing the remodeling of nascent CL by transferring fatty acids to monolyso-CL from other phospholipids (Schlame et al., 2012). The result is highly unsaturated forms of CL as represented by one of the structures found in yeast and mammalian cells. The (*) in red indicates the three chiral centers of naturally occurring CLs; carbon of the central glycerol is only a chiral center if the two adjacent phosphatidyl moieties have different fatty acid compositions. The glycerol backbone (green) is indicated. The lower figure depicts the hydrogen-bonding between the central free hydroxyl of CL and the phosphate residues creating a proton sink in the lipid bilayer and raising the pKa of one phosphate near neutrality (Haines, 2009). DAG denotes the diacylglycerol lipid domain.

Figure 2

Pseudo-atomic model shows the arrangement of CI, CIII and CIV in the bovine respirasome (PDB 2YBB, (Althoff et al., 2011)). (A) Side view and (B) view from IMS. Two CL molecules (in yellow) in the external cavity formed by cytochrome c₁, cytochrome b and closed by chain G in each monomer of CIII (PDB 1PP9) are shown. MA denotes the mitochondrial matrix.

Figure 3

Pseudo-atomic model for the yeast tetrameric SC III₂IV₂. Side view (large structure) and view from the IMS (insert) showing the arrangement of CIII (PDB 3CX2) and two CIVs (PDB 1OCC) in the SC. One CL molecule (in the external cavity of CIII), cytochrome c₁, cytochrome b and Qcr8 subunit (chain H, homolog of chain G in bovine) are colored in each CIII monomer as indicated. Derived from the structure reported in (Mileykovskaya et al., 2012).

Figure 4

CL binding sites of bovine and yeast CIII extracted from CGMD simulation of the complexes embedded in a CL/PC bilayer. CLs bound in the sites I and VI/VIa correspond to tightly bound CLs found in the crystal structures of CIII from yeast and bovine. Site I corresponds to the CL binding site in the external cavity closed by chain H in yeast and its homolog chain G in bovine. Sites VI/VIa corresponds to the conserved site for tightly bound CL in the inner cavity located close to the CIII homodimer interface (CN5, PDB 3CX5 for yeast and CL3 in PDB 1SQP for bovine). Chain K is present in the bovine CIII close to sites VI and VIa. However, there is no homolog for this chain in yeast CIII. Sites II, III, IV and V are the sites on the membrane-exposed surfaces of CIII. The figure was adapted with permission from (Arnarez et al., 2013b). Copyright (2013) American Chemical Society.