Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

Conserved cardiolipin-mitochondrial ADP/ATP carrier interactions assume distinct structural and functional roles that are clinically relevant.

The mitochondrial phospholipid cardiolipin (CL) promotes bioenergetics via oxidative phosphorylation (OXPHOS). Three tightly bound CLs are evolutionarily conserved in the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) which resides in the inner mitochondrial membrane and exchanges ADP and ATP to enable OXPHOS. Here, we investigated the role of these buried CLs in the carrier using yeast Aac2 as a model. We introduced negatively charged mutations into each CL-binding site of Aac2 to disrupt the CL interactions via electrostatic repulsion. While all mutations disturbing the CL-protein interaction destabilized Aac2 monomeric structure, transport activity was impaired in a pocket-specific manner. Finally, we determined that a disease-associated missense mutation in one CL-binding site in ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast.

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.

Deacylation on the matrix side of the mitochondrial inner membrane regulates cardiolipin remodeling.

The mitochondrial-specific lipid cardiolipin (CL) is required for numerous processes therein. After its synthesis on the matrix-facing leaflet of the inner membrane (IM), CL undergoes acyl chain remodeling to achieve its final form. In yeast, this process is completed by the transacylase tafazzin, which associates with intermembrane space (IMS)-facing membrane leaflets. Mutations in TAZ1 result in the X-linked cardiomyopathy Barth syndrome. Amazingly, despite this clear pathophysiological association, the physiological importance of CL remodeling is unresolved. In this paper, we show that the lipase initiating CL remodeling, Cld1p, is associated with the matrix-facing leaflet of the mitochondrial IM. Thus monolysocardiolipin generated by Cld1p must be transported to IMS-facing membrane leaflets to gain access to tafazzin, identifying a previously unknown step required for CL remodeling. Additionally, we show that Cld1p is the major site of regulation in CL remodeling; and that, like CL biosynthesis, CL remodeling is augmented in growth conditions requiring mitochondrially produced energy. However, unlike CL biosynthesis, dissipation of the mitochondrial membrane potential stimulates CL remodeling, identifying a novel feedback mechanism linking CL remodeling to oxidative phosphorylation capacity.

Seven functional classes of Barth syndrome mutation.

Patients with Barth syndrome (BTHS), a rare X-linked disease, suffer from skeletal and cardiomyopathy and bouts of cyclic neutropenia. The causative gene encodes tafazzin, a transacylase, which is the major determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, CL. In addition to numerous frame shift and splice-site mutations, 36 missense mutations have been associated with BTHS. Previously, we established a BTHS-mutant panel in the yeast Saccharomyces cerevisiae that successfully models 18/21 conserved pathogenic missense mutations and defined the loss-of-function mechanism associated with a subset of the mutant tafazzins. Here, we report the biochemical and cell biological characterization of the rest of the yeast BTHS-mutant panel and in so doing identify three additional modes of tafazzin dysfunction. The largest group of mutant tafazzins is catalytically null, two mutants encode hypomorphic alleles, and another two mutants are temperature sensitive. Additionally, we have expanded the defects associated with previously characterized matrix-mislocalized-mutant tafazzins to include the rapid degradation of aggregation-prone polypeptides that correctly localize to the mitochondrial IMS. In sum, our in-depth characterization of the yeast BTHS-mutant panel has identified seven functional classes of BTHS mutation.

Barth syndrome mutations that cause tafazzin complex lability.

Deficits in mitochondrial function result in many human diseases. The X-linked disease Barth syndrome (BTHS) is caused by mutations in the tafazzin gene TAZ1. Its product, Taz1p, participates in the metabolism of cardiolipin, the signature phospholipid of mitochondria. In this paper, a yeast BTHS mutant tafazzin panel is established, and 18 of the 21 tested BTHS missense mutations cannot functionally replace endogenous tafazzin. Four BTHS mutant tafazzins expressed at low levels are degraded by the intermembrane space AAA (i-AAA) protease, suggesting misfolding of the mutant polypeptides. Paradoxically, each of these mutant tafazzins assembles in normal protein complexes. Furthermore, in the absence of the i-AAA protease, increased expression and assembly of two of the BTHS mutants improve their function. However, the BTHS mutant complexes are extremely unstable and accumulate as insoluble aggregates when disassembled in the absence of the i-AAA protease. Thus, the loss of function for these BTHS mutants results from the inherent instability of the mutant tafazzin complexes.