Altered Lipid Synthesis by Lack of Yeast Pah1 Phosphatidate Phosphatase Reduces Chronological Life Span.

In Saccharomyces cerevisiae, Pah1 phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, plays a crucial role in the synthesis of the storage lipid triacylglycerol. This evolutionarily conserved enzyme also plays a negative regulatory role in controlling de novo membrane phospholipid synthesis through its consumption of phosphatidate. We found that the pah1Δ mutant was defective in the utilization of non-fermentable carbon sources but not in oxidative phosphorylation; the mutant did not exhibit major changes in oxygen consumption rate, mitochondrial membrane potential, F1F0-ATP synthase activity, or gross mitochondrial morphology. The pah1Δ mutant contained an almost normal complement of major mitochondrial phospholipids with some alterations in molecular species. Although oxidative phosphorylation was not compromised in the pah1Δ mutant, the cellular levels of ATP in quiescent cells were reduced by 2-fold, inversely correlating with a 4-fold increase in membrane phospholipids. In addition, the quiescent pah1Δ mutant cells had 3-fold higher levels of mitochondrial superoxide and cellular lipid hydroperoxides, had reduced activities of superoxide dismutase 2 and catalase, and were hypersensitive to hydrogen peroxide. Consequently, the pah1Δ mutant had a shortened chronological life span. In addition, the loss of Tsa1 thioredoxin peroxidase caused a synthetic growth defect with the pah1Δ mutation. The shortened chronological life span of the pah1Δ mutant along with its growth defect on non-fermentable carbon sources and hypersensitivity to hydrogen peroxide was suppressed by the loss of Dgk1 diacylglycerol kinase, indicating that the underpinning of pah1Δ mutant defects was the excess synthesis of membrane phospholipids.

Cardiolipin-dependent reconstitution of respiratory supercomplexes from purified Saccharomyces cerevisiae complexes III and IV.

Here, we report for the first time in vitro reconstitution of the respiratory supercomplexes from individual complexes III and IV. Complexes III and IV were purified from Saccharomyces cerevisiae mitochondria. Complex III contained eight molecules of cardiolipin, and complex IV contained two molecules of cardiolipin, as determined by electrospray ionization-mass spectrometry. Complex IV also contained Rcf1p. No supercomplexes were formed upon mixing of the purified complexes, and low amounts of the supercomplex trimer III(2)IV(1) were formed after reconstitution into proteoliposomes containing only phosphatidylcholine and phosphatidylethanolamine. Further addition of cardiolipin to the proteoliposome reconstitution mixture resulted in distinct formation of both the III(2)IV(1) supercomplex trimer and III(2)IV(2) supercomplex tetramer. No other anionic phospholipid was as effective as cardiolipin in supporting tetramer formation. Phospholipase treatment of complex IV prevented trimer formation in the absence of cardiolipin. Both trimer and tetramer formations were restored by cardiolipin. Analysis of the reconstituted tetramer by single particle electron microscopy confirmed native organization of individual complexes within the supercomplex. In conclusion, although some trimer formation occurred dependent only on tightly bound cardiolipin, tetramer formation required additional cardiolipin. This is consistent with the high cardiolipin content in the native tetramer. The dependence on cardiolipin for supercomplex formation suggests that changes in cardiolipin levels resulting from changes in physiological conditions may control the equilibrium between individual respiratory complexes and supercomplexes in vivo.

Arrangement of the respiratory chain complexes in Saccharomyces cerevisiae supercomplex III2IV2 revealed by single particle cryo-electron microscopy.

Here we present for the first time a three-dimensional cryo-EM map of the Saccharomyces cerevisiae respiratory supercomplex composed of dimeric complex III flanked on each side by one monomeric complex IV. A precise fit of the existing atomic x-ray structures of complex III from yeast and complex IV from bovine heart into the cryo-EM map resulted in a pseudo-atomic model of the three-dimensional structure for the supercomplex. The distance between cytochrome c binding sites of complexes III and IV is about 6 nm, which supports proposed channeling of cytochrome c between the individual complexes. The opposing surfaces of complexes III and IV differ considerably from those reported for the bovine heart supercomplex as determined by cryo-EM. A closer association between the individual complex domains at the aqueous membrane interface and larger spaces between the membrane-embedded domains where lipid molecules may reside are also demonstrated. The supercomplex contains about 50 molecules of cardiolipin (CL) with a fatty acid composition identical to that of the inner membrane CL pool, consistent with CL-dependent stabilization of the supercomplex.

Molecular Basis of Cell Membrane Adaptation in Daptomycin-Resistant Enterococcus faecalis.

Daptomycin is a last-resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by re-distributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a three-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the two genes ( cls1 and cls2 ) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, as an important mediator of CM remodeling required for re-distribution of anionic phospholipid microdomains via interactions with Cls1. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.

Structural insights into cardiolipin replacement by phosphatidylglycerol in a cardiolipin-lacking yeast respiratory supercomplex.

Cardiolipin is a hallmark phospholipid of mitochondrial membranes. Despite established significance of cardiolipin in supporting respiratory supercomplex organization, a mechanistic understanding of this lipid-protein interaction is still lacking. To address the essential role of cardiolipin in supercomplex organization, we report cryo-EM structures of a wild type supercomplex (IV1III2IV1) and a supercomplex (III2IV1) isolated from a cardiolipin-lacking Saccharomyces cerevisiae mutant at 3.2-Å and 3.3-Å resolution, respectively, and demonstrate that phosphatidylglycerol in III2IV1 occupies similar positions as cardiolipin in IV1III2IV1. Lipid-protein interactions within these complexes differ, which conceivably underlies the reduced level of IV1III2IV1 and high levels of III2IV1 and free III2 and IV in mutant mitochondria. Here we show that anionic phospholipids interact with positive amino acids and appear to nucleate a phospholipid domain at the interface between the individual complexes, which dampen charge repulsion and further stabilize interaction, respectively, between individual complexes.

Bacterial cell membranes and their role in daptomycin resistance: A review.

Lipids play a major role in bacterial cells. Foremost, lipids are the primary constituents of the cell membrane bilayer, providing structure and separating the cell from the surrounding environment. This makes the lipid bilayer a prime target for antimicrobial peptides and membrane-acting antibiotics such as daptomycin. In response, bacteria have evolved mechanisms by which the membrane can be adapted to resist attack by these antimicrobial compounds. In this review, we focus on the membrane phospholipid changes associated with daptomycin resistance in enterococci, Staphylococcus aureus, and the Viridans group streptococci.

Functional taxonomy of bacterial hyperstructures.

The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures. Here, we review a variety of spatially extended structures, complexes, and assemblies that might be termed hyperstructures. These include ribosomal or "nucleolar" hyperstructures; transertion hyperstructures; putative phosphotransferase system and glycolytic hyperstructures; chemosignaling and flagellar hyperstructures; DNA repair hyperstructures; cytoskeletal hyperstructures based on EF-Tu, FtsZ, and MreB; and cell cycle hyperstructures responsible for DNA replication, sequestration of newly replicated origins, segregation, compaction, and division. We propose principles for classifying these hyperstructures and finally illustrate how thinking in terms of hyperstructures may lead to a different vision of the bacterial cell.

Cardiolipin membrane domains in prokaryotes and eukaryotes.

Cardiolipin (CL) plays a key role in dynamic organization of bacterial and mitochondrial membranes. CL forms membrane domains in bacterial cells, and these domains appear to participate in binding and functional regulation of multi-protein complexes involved in diverse cellular functions including cell division, energy metabolism, and membrane transport. Visualization of CL domains in bacterial cells by the fluorescent dye 10-N-nonyl acridine orange is critically reviewed. Possible mechanisms proposed for CL dynamic localization in bacterial cells are discussed. In the mitochondrial membrane CL is involved in organization of multi-subunit oxidative phosphorylation complexes and in their association into higher order supercomplexes. Evidence suggesting a possible role for CL in concert with ATP synthase oligomers in establishing mitochondrial cristae morphology is presented. Hypotheses on CL-dependent dynamic re-organization of the respiratory chain in response to changes in metabolic states and CL dynamic re-localization in mitochondria during the apoptotic response are briefly addressed.

Cardiolipin Synthesis in Skeletal Muscle Is Rhythmic and Modifiable by Age and Diet.

Circadian clocks regulate metabolic processes in a tissue-specific manner, which deteriorates during aging. Skeletal muscle is the largest metabolic organ in our body, and our previous studies highlight a key role of circadian regulation of skeletal muscle mitochondria in healthy aging. However, a possible circadian regulation of cardiolipin (CL), the signature lipid class in the mitochondrial inner membrane, remains largely unclear. Here, we show that CL levels oscillate during the diurnal cycle in C2C12 myotubes. Disruption of the Ror genes, encoding the ROR nuclear receptors in the secondary loop of the circadian oscillator, in C2C12 cells was found to dampen core circadian gene expression. Importantly, several genes involved in CL synthesis, including Taz and Ptpmt1, displayed rhythmic expression which was disrupted or diminished in Ror-deficient C2C12 cells. In vivo studies using skeletal muscle tissues collected from young and aged mice showed diverse effects of the clock and aging on the oscillatory expression of CL genes, and CL levels in skeletal muscle were enhanced in aged mice relative to young mice. Finally, consistent with a regulatory role of RORs, Nobiletin, a natural agonist of RORs, was found to partially restore transcripts levels of CL synthesis genes in aged muscle under a dietary challenge condition. Together, these observations highlight a rhythmic CL synthesis in skeletal muscle that is dependent on RORs and modifiable by age and diet.

Mutual effects of MinD-membrane interaction: II. Domain structure of the membrane enhances MinD binding.

MinD, a well-conserved bacterial amphitropic protein involved in spatial regulation of cell division, has a typical feature of reversible binding to the membrane. MinD shows a clear preference for acidic phospholipids organized into lipid domains in bacterial membrane. We have shown that binding of MinD may change the dynamics of model and native membranes (see accompanying paper [1]). On the other hand, MinD dimerization and anchoring could be enhanced on pre-existing anionic phospholipid domains. We have tested MinD binding to model membranes in which acidic and zwitterionic phospholipids are either well-mixed or segregated to phase domains. The phase separation was achieved in binary mixtures of 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol] (SOPG) with 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC) or 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG) and binding to these membranes was compared with that to a fluid mixture of SOPG with 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC). The results demonstrate that MinD binding to the membrane is enhanced by segregation of anionic phospholipids to fluid domains in a gel-phase environment and, moreover, the protein stabilizes such domains. This suggests that an uneven binding of MinD to the heterogeneous native membrane is possible, leading to formation of a lipid-specific distribution pattern of MinD and/or modulation of its temporal behavior.

Mutual effects of MinD-membrane interaction: I. Changes in the membrane properties induced by MinD binding.

In Escherichia coli and other bacteria, MinD, along with MinE and MinC, rapidly oscillates from one pole of the cell to the other controlling the correct placement of the division septum. MinD binds to the membrane through its amphipathic C-terminal alpha-helix. This binding, promoted by ATP-induced dimerization, may be further enhanced by a consequent attraction of acidic phospholipids and formation of a stable proteolipid domain. In the context of this hypothesis we studied changes in dynamics of a model membrane caused by MinD binding using membrane-embedded fluorescent probes as reporters. A remarkable increase in membrane viscosity and order upon MinD binding to acidic phospholipids was evident from the pyrene and DPH fluorescence changes. This viscosity increase is cooperative with regards to the concentration of MinD-ATP, but not of the ADP form, indicative of dimerization. Moreover, similar changes in the membrane dynamics were demonstrated in the native inverted cytoplasmic membranes of E. coli, with a different depth effect. The mobility of pyrene-labeled phosphatidylglycerol indicated formation of acidic phospholipid-enriched domains in a mixed acidic-zwitterionic membrane at specific MinD/phospholipid ratios. A comparison between MinD from E. coli and Neisseria gonorrhea is also presented.

Lipids in the assembly of membrane proteins and organization of protein supercomplexes: implications for lipid-linked disorders.

Lipids play important roles in cellular dysfunction leading to disease. Although a major role for phospholipids is in defining the membrane permeability barrier, phospholipids play a central role in a diverse range of cellular processes and therefore are important factors in cellular dysfunction and disease. This review is focused on the role of phospholipids in normal assembly and organization of the membrane proteins, multimeric protein complexes, and higher order supercomplexes. Since lipids have no catalytic activity, it is difficult to determine their function at the molecular level. Lipid function has generally been defined by affects on protein function or cellular processes. Molecular details derived from genetic, biochemical, and structural approaches are presented for involvement of phosphatidylethanolamine and cardiolipin in protein organization. Experimental evidence is presented that changes in phosphatidylethanolamine levels results in misfolding and topological misorientation of membrane proteins leading to dysfunctional proteins. Examples are presented for diseases in which proper protein folding or topological organization is not attained due to either demonstrated or proposed involvement of a lipid. Similar changes in cardiolipin levels affects the structure and function of individual components of the mitochondrial electron transport chain and their organization into supercomplexes resulting in reduced mitochondrial oxidative phosphorylation efficiency and apoptosis. Diseases in which mitochondrial dysfunction has been linked to reduced cardiolipin levels are described. Therefore, understanding the principles governing lipid-dependent assembly and organization of membrane proteins and protein complexes will be useful in developing novel therapeutic approaches for disorders in which lipids play an important role.

Nobiletin fortifies mitochondrial respiration in skeletal muscle to promote healthy aging against metabolic challenge.

Circadian disruption aggravates age-related decline and mortality. However, it remains unclear whether circadian enhancement can retard aging in mammals. We previously reported that the small molecule Nobiletin (NOB) activates ROR (retinoid acid receptor-related orphan receptor) nuclear receptors to potentiate circadian oscillation and protect against metabolic dysfunctions. Here we show that NOB significantly improves metabolic fitness in naturally aged mice fed with a regular diet (RD). Furthermore, NOB enhances healthy aging in mice fed with a high-fat diet (HF). In HF skeletal muscle, the NOB-ROR axis broadly activates genes for mitochondrial respiratory chain complexes (MRCs) and fortifies MRC activity and architecture, including Complex II activation and supercomplex formation. These mechanisms coordinately lead to a dichotomous mitochondrial optimization, namely increased ATP production and reduced ROS levels. Together, our study illustrates a focal mechanism by a clock-targeting pharmacological agent to optimize skeletal muscle mitochondrial respiration and promote healthy aging in metabolically stressed mammals.

Behaviour of bacterial division protein FtsZ under a monolayer with phospholipid domains.

Assembly of the tubulin-like protein FtsZ at or near the cytoplasmic membrane is one of the earliest steps in division of bacteria such as Escherichia coli. Exactly what constitutes the site at which FtsZ acts is less clear. To investigate the influence of the membrane phospholipids on FtsZ localization and assembly, we have elaborated with the Langmuir technique a two-lipid monolayer made of dilauryl-phosphatidylethanolamine (DLPE) and dipalmitoyl-phosphatidylglycerol (DPPG). This monolayer comprised stable condensed domains in an expanded continuous phase. In the presence of GTP, FtsZ assembly disrupts the condensed domains within 5 min. After several hours, with or without GTP, FtsZ assembled into large aggregates at the domain interface. We suggest that the GTP-induced polymerization of FtsZ is coupled to the association of FtsZ protofilaments with domain interfaces.

Role of membrane lipids in bacterial division-site selection.

Heterogeneous distribution of specific phospholipids along the bacterial membrane results in the formation of domains enriched in anionic phospholipids at the cell poles and cell center, which appear to participate in the binding of amphitropic proteins responsible for selection and recognition of the division site. It was discovered that functioning of the Min system, which protects the cell poles from aberrant positioning of the Z-ring, is controlled by direct interaction of its MinD component with membrane phospholipids. There is also an accumulation of evidence that the mid-cell domain, formed in the cell at a defined step of the cell cycle, provides the optimal phospholipid composition first for initiation of DNA replication and then for Z-ring positioning.

Diversity and versatility of lipid-protein interactions revealed by molecular genetic approaches.

The diversity in structures and physical properties of lipids provides a wide variety of possible interactions with proteins that affect their assembly, organization, and function either at the surface of or within membranes. Because lipids have no catalytic activity, it has been challenging to define many of their precise functions in vivo in molecular terms. Those processes responsive to lipids are attuned to the native lipid environment for optimal function, but evidence that lipids with similar properties or even detergents can sometimes partially replace the natural lipid environment has led to uncertainty as to the requirement for specific lipids. The development of strains of microorganisms in which membrane lipid composition can be genetically manipulated in viable cells has provided a set of reagents to probe lipid functions. These mutants have uncovered previously unrecognized roles for lipids and provided in vivo verification for putative functions described in vitro. In this review, we summarize how these reagent strains have provided new insight into the function of lipids. The role of specific lipids in membrane protein folding and topological organization is reviewed. The evidence is summarized for the involvement of anionic lipid-enriched domains in the organization of amphitropic proteins on the membrane surface into molecular machines involved in DNA replication and cell division.

The membrane: transertion as an organizing principle in membrane heterogeneity.

The bacterial membrane exhibits a significantly heterogeneous distribution of lipids and proteins. This heterogeneity results mainly from lipid-lipid, protein-protein, and lipid-protein associations which are orchestrated by the coupled transcription, translation and insertion of nascent proteins into and through membrane (transertion). Transertion is central not only to the individual assembly and disassembly of large physically linked groups of macromolecules (alias hyperstructures) but also to the interactions between these hyperstructures. We review here these interactions in the context of the processes in Bacillus subtilis and Escherichia coli of nutrient sensing, membrane synthesis, cytoskeletal dynamics, DNA replication, chromosome segregation, and cell division.

A hypothesis to explain division site selection in Escherichia coli by combining nucleoid occlusion and Min.

The positioning of the site of cell division in Escherichia coli results, it is generally believed, from the operation of nucleoid occlusion in combination with the Min system. Nucleoid occlusion prevents division over the nucleoids and directs it by default to the mid-cell region between segregating nucleoids or to polar regions while the Min system prevents division in polar regions. Unresolved questions include how these systems interact to control the earliest known event in division, the assembly at the membrane of the tubulin-like protein, FtsZ, and, more importantly, what exactly constitutes a division site. Evidence exists that (1) the coupled transcription, translation and insertion of proteins into membrane (transertion), can structure the cytoplasmic membrane into phospholipid domains, (2) the MinD protein can convert vesicles into tubes and (3) a variety of membranous structures can be observed at mid-cell. These data support a model in which transertion from the segregating daughter chromosomes leads to the formation of a distinct proteolipid domain between them at mid-cell; the composition of this domain allows phospholipid tubes to extend like fingers into the cytoplasm; these tubes then become the substrate for the dynamic assembly and disassembly of FtsZ which converts them into the invaginating fold responsible for division; the Min system inhibits division at unwanted sites and times by removing these tubes especially at the cell poles.

Cardiolipin-dependent formation of mitochondrial respiratory supercomplexes.

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.

N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill bacteria by interacting with bacterial membrane lipids but only N-acylated peptides interact with both charged cardiolipin and zwitterionic phosphatidylethanolamine resulting in remodeling of the natural phospholipid domains in the E. coli membrane that leads to defects in cell division.