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.

A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling.

Membrane protein recycling systems are essential for maintenance of the endosome-lysosome system. In yeast, retromer and Snx4 coat complexes are recruited to the endosomal surface, where they recognize cargos. They sort cargo and deform the membrane into recycling tubules that bud from the endosome and target to the Golgi. Here, we reveal that the SNX-BAR protein, Mvp1, mediates an endosomal recycling pathway that is mechanistically distinct from the retromer and Snx4 pathways. Mvp1 deforms the endosomal membrane and sorts cargos containing a specific sorting motif into a membrane tubule. Subsequently, Mvp1 recruits the dynamin-like GTPase Vps1 to catalyze membrane scission and release of the recycling tubule. Similarly, SNX8, the human homolog of Mvp1, which has been also implicated in Alzheimer's disease, mediates formation of an endosomal recycling tubule. Thus, we present evidence for a novel endosomal retrieval pathway that is conserved from yeast to humans.

Activity of a ubiquitin ligase adaptor is regulated by disordered insertions in its arrestin domain.

The protein composition of the plasma membrane is rapidly remodeled in response to changes in nutrient availability or cellular stress. This occurs, in part, through the selective ubiquitylation and endocytosis of plasma membrane proteins, which in the yeast Saccharomyces cerevisiae is mediated by the HECT E3 ubiquitin ligase Rsp5 and arrestin--related trafficking (ART) adaptors. Here, we provide evidence that the ART protein family members are composed of an arrestin fold with interspersed disordered loops. Using Art1 as a model, we show that these loop and tail regions, while not strictly required for function, regulate its activity through two separate mechanisms. Disruption of one loop mediates Art1 substrate specificity. Other loops are subjected to phosphorylation in a manner dependent on the Pho85 cyclins Clg1 and Pho80. Phosphorylation of the loops controls Art1's localization to the plasma membrane, which promotes cargo ubiquitylation and endocytosis, demonstrating a mechanism through which Art1 activity is regulated.

Methods for studying the regulation of membrane traffic by ubiquitin and the ESCRT pathway.

Covalent modification of proteins with ubiquitin dynamically regulates their function and fate. The ubiquitination of most plasma membrane proteins initiates endocytosis and ESCRT-mediated sorting to the lysosomal lumen for degradation. Powerful genetic approaches in the budding yeast Saccharomyces cerevisiae have been particularly instrumental in the discovery and elucidation of these molecular mechanisms, which are conserved in all eukaryotes. Here we provide two detailed protocols and tools for studying ubiquitination-dependent membrane trafficking mechanisms in yeast. The first utilizes fusions between a protein of interest and an auxotrophic marker to screen for mutants that affect ubiquitin-mediated endocytosis. The second method artificially ubiquitinates a protein of interest, allowing downstream trafficking steps to be studied independently from the regulatory signals that initiate endocytosis.

Cardiomyopathy-associated mutation in the ADP/ATP carrier reveals translation-dependent regulation of cytochrome c oxidase activity.

How the absence of the major mitochondrial ADP/ATP carrier in yeast, Aac2p, results in a specific defect in cytochrome c oxidase (COX; complex IV) activity is a long-standing mystery. Aac2p physically associates with respiratory supercomplexes, which include complex IV, raising the possibility that its activity is dependent on its association with Aac2p. Here, we have leveraged a transport-dead pathogenic AAC2 point mutant to determine the basis for the reduced COX activity in the absence of Aac2p. The steady-state levels of complex IV subunits encoded by the mitochondrial genome are significantly reduced in the absence of Aac2p function, whether its association with respiratory supercomplexes is preserved or not. This diminution in COX amounts is not caused by a reduction in the mitochondrial genome copy number or the steady-state level of its transcripts, and does not reflect a defect in complex IV assembly. Instead, the absence of Aac2p activity, genetically or pharmacologically, results in an aberrant pattern of mitochondrial translation. Interestingly, compared with the complete absence of Aac2p, the complex IV-related defects are greater in mitochondria expressing the transport-inactive Aac2p mutant. Our results highlight a critical role for Aac2p transport in mitochondrial translation whose disturbance uniquely impacts cytochrome c oxidase.

Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.

In yeast, phosphatidylglycerol (PG) is a minor phospholipid under standard conditions; it can be utilized for cardiolipin (CL) biosynthesis by CL synthase, Crd1p, or alternatively degraded by the phospholipase Pgc1p. The Saccharomyces cerevisiae deletion mutants crd1Δ and pgc1Δ both accumulate PG. Based on analyses of the phospholipid content of pgc1Δ and crd1Δ yeast, we revealed that in yeast mitochondria, two separate pools of PG are present, which differ in their fatty acid composition and accessibility for Pgc1p-catalyzed degradation. In contrast to CL-deficient crd1Δ yeast, the pgc1Δ mutant contains normal levels of CL. This makes the pgc1Δ strain a suitable model to study the effect of accumulation of PG per se. Using fluorescence microscopy, we show that accumulation of PG with normal levels of CL resulted in increased fragmentation of mitochondria, while in the absence of CL, accumulation of PG led to the formation of large mitochondrial sheets. We also show that pgc1Δ mitochondria exhibited increased respiration rates due to increased activity of cytochrome c oxidase. Taken together, our results indicate that not only a lack of anionic phospholipids, but also excess PG, or unbalanced ratios of anionic phospholipids in mitochondrial membranes, have harmful consequences on mitochondrial morphology and function.

The topology and regulation of cardiolipin biosynthesis and remodeling in yeast.

The signature mitochondrial phospholipid cardiolipin plays an important role in mitochondrial function, and alterations in cardiolipin metabolism are associated with human disease. Topologically, cardiolipin biosynthesis and remodeling are complex. Precursor phospholipids must be transported from the ER, across the mitochondrial outer membrane to the matrix-facing leaflet of the inner membrane, where cardiolipin biosynthesis commences. Post-synthesis, cardiolipin undergoes acyl chain remodeling, requiring additional trafficking steps, before it achieves its final distribution within both mitochondrial membranes. This process is regulated at several points via multiple independent mechanisms. Here, we review the regulation and topology of cardiolipin biosynthesis and remodeling in the yeast Saccharomyces cerevisiae. Although cardiolipin metabolism is more complicated in mammals, yeast have been an invaluable model for dissecting the steps required for this process.

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.

The power of yeast to model diseases of the powerhouse of the cell.

Mitochondria participate in a variety of cellular functions. As such, mitochondrial diseases exhibit numerous clinical phenotypes. Because mitochondrial functions are highly conserved between humans and Saccharomyces cerevisiae, yeast are an excellent model to study mitochondrial disease, providing insight into both physiological and pathophysiological processes.

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.