Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Dopey proteins are essential but overlooked regulators of membrane trafficking.

Dopey family proteins play crucial roles in diverse processes from morphogenesis to neural function and are conserved from yeast to mammals. Understanding the mechanisms behind these critical functions could have major clinical significance, as dysregulation of Dopey proteins has been linked to the cognitive defects in Down syndrome, as well as neurological diseases. Dopey proteins form a complex with the non-essential GEF-like protein Mon2 and an essential lipid flippase from the P4-ATPase family. Different combinations of Dopey, Mon2 and flippases have been linked to regulating membrane remodeling, from endosomal recycling to extracellular vesicle formation, through their interactions with lipids and other membrane trafficking regulators, such as ARL1, SNX3 and the kinesin-1 light chain KLC2. Despite these important functions and their likely clinical significance, Dopey proteins remain understudied and their roles elusive. Here, we review the major scientific discoveries relating to Dopey proteins and detail key open questions regarding their function to draw attention to these fascinating enigmas.

MlaC belongs to a unique class of non-canonical substrate-binding proteins and follows a novel phospholipid-binding mechanism.

The outer membrane (OM) of Gram-negative bacteria acts as a formidable barrier against a plethora of detrimental compounds owing to its asymmetric nature. This is because the OM possesses lipopolysaccharides (LPSs) in the outer leaflet and phospholipids (PLs) in the inner leaflet. The maintenance of lipid asymmetry (Mla) system is involved in preserving the distribution of PLs in OM. The periplasmic component of the system MlaC serves as the substrate-binding protein (SBP) that shuttles PLs between the inner and outer membranes. However, an in-depth report highlighting its mechanism of ligand binding is still lacking. This study reports the crystal structure of MlaC from Escherichia coli (EcMlaC) at a resolution of 2.5 Å in a quasi-open state, complexed with PL. The structural analysis reveals that EcMlaC and orthologs comprise two major domains, viz. nuclear transport factor 2-like (NTF2-like) and phospholipid-binding protein (PBP). Each domain can be further divided into two subdomains arranged in a discontinuous fashion. This study further reveals that EcMlaC is polyspecific in nature and follows a reverse mechanism of the opening of the substrate-binding site during the ligand binding. Furthermore, MlaC can bind two PLs by forming subsites in the binding pocket. These findings, altogether, have led to the proposition of the unique "segmented domain movement" mechanism of PL binding, not reported for any known SBP to date. Further, unlike typical SBPs, MlaC has originated from a cystatin-like fold. Overall, this study establishes MlaC to be a non-canonical SBP with a unique ligand-binding mechanism.

Regulatory Roles of N- and C-Terminal Cytoplasmic Regions of P4-ATPases.

P4-ATPases, which are subfamily members of P-type ATPase superfamily, translocate membrane lipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet, thus regulating trans-bilayer lipid asymmetry. Mammalian P4-ATPases localize to the specific subcellular organelles or the plasma membrane where they translocate the specific lipids. Although recent advances in the structural analysis of P4-ATPases have improved our understanding of lipid transporting machinery, the mechanism of substrate specificity and the regulatory mechanism of the enzymes remain largely unknown. Recent studies have uncovered several specific localization and regulatory mechanisms of P4-ATPases. Here, we review the current understanding of the regulatory mechanism of P4-ATPase activity and localization in mammalian cells.

The Rim101 pathway mediates adaptation to external alkalization and altered lipid asymmetry: hypothesis describing the detection of distinct stresses by the Rim21 sensor protein.

Yeast cells adapt to alkaline conditions by activating the Rim101 alkali-responsive pathway. Rim21 acts as a sensor in the Rim101 pathway and detects extracellular alkalization. Interestingly, Rim21 is also known to be activated by alterations involving the lipid asymmetry of the plasma membrane. In this study, we briefly summarize the mechanism of activation and the signal transduction cascade of the Rim101 pathway and propose a hypothesis on how Rim21 is able to detect distinct signals, particularly external alkalization, and altered lipid asymmetry. We found that external alkalization can suppress transbilayer movements of phospholipids between the two leaflets of the plasma membrane, which may lead to the disturbance of the lipid asymmetry of the plasma membrane. Therefore, we propose that external alteration is at least partly sensed by Rim21 through alterations in lipid asymmetry. Understanding this activation mechanism could greatly contribute to drug development against fungal infections.

Cholesterol asymmetry at the tip of filopodia during cell adhesion.

During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation.

Fluorescence sensors for imaging membrane lipid domains and cholesterol.

Lipid membrane domains are supramolecular lateral heterogeneities of biological membranes. Of nanoscopic dimensions, they constitute specialized hubs used by the cell as transient signaling platforms for a great variety of biologically important mechanisms. Their property to form and dissolve in the bulk lipid bilayer endow them with the ability to engage in highly dynamic processes, and temporarily recruit subpopulations of membrane proteins in reduced nanometric compartments that can coalesce to form larger mesoscale assemblies. Cholesterol is an essential component of these lipid domains; its unique molecular structure is suitable for interacting intricately with crevices and cavities of transmembrane protein surfaces through its rough ß face while "talking" to fatty acid acyl chains of glycerophospholipids and sphingolipids via its smooth α face. Progress in the field of membrane domains has been closely associated with innovative improvements in fluorescence microscopy and new fluorescence sensors. These advances enabled the exploration of the biophysical properties of lipids and their supramolecular platforms. Here I review the rationale behind the use of biosensors over the last few decades and their contributions towards elucidation of the in-plane and transbilayer topography of cholesterol-enriched lipid domains and their molecular constituents. The challenges introduced by super-resolution optical microscopy are discussed, as well as possible scenarios for future developments in the field, including virtual ("no staining") staining.

Extracellular vesicle budding is inhibited by redundant regulators of TAT-5 flippase localization and phospholipid asymmetry.

Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.

N-glycosylation of Rim21 at an Unconventional Site Fine-tunes Its Behavior in the Plasma Membrane.

The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.

Lipid trafficking across the Gram-negative cell envelope.

The outer membrane (OM) of Gram-negative bacteria exhibits unique lipid asymmetry, with lipopolysaccharides (LPS) residing in the outer leaflet and phospholipids (PLs) in the inner leaflet. This asymmetric bilayer protects the bacterium against intrusion of many toxic substances, including antibiotics and detergents, yet allows acquisition of nutrients necessary for growth. To build the OM and ensure its proper function, the cell produces OM constituents in the cytoplasm or inner membrane and transports these components across the aqueous periplasmic space separating the two membranes. Of note, the processes by which the most basic membrane building blocks, i.e. PLs, are shuttled across the cell envelope remain elusive. This review highlights our current understanding (or lack thereof) of bacterial PL trafficking, with a focus on recent developments in the field. We adopt a mechanistic approach and draw parallels and comparisons with well-characterized systems, particularly OM lipoprotein and LPS transport, to illustrate key challenges in intermembrane lipid trafficking. Pathways that transport PLs across the bacterial cell envelope are fundamental to OM biogenesis and homeostasis and are potential molecular targets that could be exploited for antibiotic development.

The architecture of the OmpC-MlaA complex sheds light on the maintenance of outer membrane lipid asymmetry in Escherichia coli.

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, including antibiotics and bile salts. In Escherichia coli, the complex comprising osmoporin OmpC and the OM lipoprotein MlaA is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How this complex performs this function is unknown. Here, we defined the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. Using in vivo photo-cross-linking and molecular dynamics simulations, we established that MlaA interacts extensively with OmpC and is located entirely within the lipid bilayer. In addition, MlaA forms a hydrophilic channel, likely enabling PL translocation across the OM. We further showed that flexibility in a hairpin loop adjacent to the channel is critical in modulating MlaA activity. Finally, we demonstrated that OmpC plays a functional role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antibacterial drug development.

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

Changes in Membrane Ceramide Pools in Rat Soleus Muscle in Response to Short-Term Disuse.

Lipid raft disruption is an early event during skeletal muscle unloading. Ceramide (Cer) serves as a signaling lipid that can contribute to lipid raft disturbance and muscle atrophy. Using biochemical and fluorescent approaches, the distribution of Cer and related molecules in the rat soleus muscle subjected to 12 h of hindlimb suspension (HS) was studied. HS led to upregulation of TNFα receptor 1 (TNFR1), Cer-producing enzymes, and acid and neutral sphingomyelinase (SMase) in detergent-resistant membranes (lipid rafts), which was accompanied by an increase in Cer and a decrease in sphingomyelin in this membrane fraction. Fluorescent labeling indicated increased Cer in the sarcoplasm as well as the junctional (synaptic) and extrajunctional compartments of the suspended muscles. Also, a loss of membrane asymmetry (a hallmark of membrane disturbance) was induced by HS. Pretreatment with clomipramine, a functional inhibitor of acid SMase, counteracted HS-mediated changes in the Cer/sphingomyelin ratio and acid SMase abundance as well as suppressed Cer accumulation in the intracellular membranes of junctional and extrajunctional regions. However, the elevation of plasma membrane Cer and disturbance of the membrane asymmetry were suppressed only in the junctional compartment. We suggest that acute HS leads to TNFR1 and SMase upregulation in the lipid raft fraction and deposition of Cer throughout the sarcolemma and intracellularly. Clomipramine-mediated downregulation of acid SMase can suppress Cer accumulation in all compartments, excluding the extrajunctional plasma membrane.

The Rim101 pathway contributes to ER stress adaptation through sensing the state of plasma membrane.

Yeast cells sense alterations in the plasma membrane (PM) lipid asymmetry and external alkalization by the sensor protein Rim21, which functions in the Rim101 pathway. Rim101 signaling is initiated at the PM by the recruitment of the Rim101 signaling complex. The PM physically associates with the cortical endoplasmic reticulum (ER) to form ER-PM contact sites, where several signaling events, lipid exchange, and ion transport take place. In the present study, we investigated the spatial relationship between ER-PM contact sites and the sites of Rim101 signaling. Rim101 signaling mostly proceeds outside ER-PM contact sites in the PM and did not require intact ER-PM contact for its activation. Rather, the Rim101 pathway was constitutively activated by ER-PM contact site disruption, which is known to cause ER stress. ER stress induced by tunicamycin treatment activated the Rim101 pathway. Furthermore, the sensitivity of cells to tunicamycin without ER-PM contact was considerably elevated by the deletion of RIM21. These results suggest that the Rim101 pathway is important for the adaptation to ER stress by compensating for alterations in PM lipid asymmetry induced by ER stress.

Effect of lipid composition and amino acid sequence upon transmembrane peptide-accelerated lipid transleaflet diffusion (flip-flop).

We examined how hydrophobic peptide-accelerated transleaflet lipid movement (flip-flop) was affected by peptide sequence and vesicle composition and properties. A peptide with a completely hydrophobic sequence had little if any effect upon flip-flop. While peptides with a somewhat less hydrophobic sequence accelerated flip-flop, the half-time remained slow (hours) with substantial (0.5mol%) peptide in the membranes. It appears that peptide-accelerated lipid flip-flop involves a rare event that may reflect a rare state of the peptide or lipid bilayer. There was no simple relationship between peptide overall hydrophobicity and flip-flop. In addition, flip-flop was not closely linked to whether the peptides were in a transmembrane or non-transmembrane (interfacial) inserted state. Flip-flop was also not associated with peptide-induced pore formation. We found that peptide-accelerated flip-flop is initially faster in small (highly curved) unilamellar vesicles relative to that in large unilamellar vesicles. Peptide-accelerated flip-flop was also affected by lipid composition, being slowed in vesicles with thick bilayers or those containing 30% cholesterol. Interestingly, these factors also slow spontaneous lipid flip-flop in the absence of peptide. Combined with previous studies, the results are most consistent with acceleration of lipid flip-flop by peptide-induced thinning of bilayer width.

The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae.

BACKGROUND: Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci. RESULTS: In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions' adaptive nature. CONCLUSIONS: The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.

Opt2 mediates the exposure of phospholipids during cellular adaptation to altered lipid asymmetry.

Plasma membrane lipid asymmetry is important for various membrane-associated functions and is regulated by membrane proteins termed flippases and floppases. The Rim101 pathway senses altered lipid asymmetry in the yeast plasma membrane. The mutant lem3Δ cells, in which lipid asymmetry is disturbed owing to the inactivation of the plasma membrane flippases, showed a severe growth defect when the Rim101 pathway was impaired. To identify factors involved in the Rim101-pathway-dependent adaptation to altered lipid asymmetry, we performed DNA microarray analysis and found that Opt2 induced by the Rim101 pathway plays an important role in the adaptation to altered lipid asymmetry. Biochemical investigation of Opt2 revealed its localization to the plasma membrane and the Golgi, and provided several lines of evidence for the Opt2-mediated exposure of phospholipids. In addition, Opt2 was found to be required for the maintenance of vacuolar morphology and polarized cell growth. These results suggest that Opt2 is a novel factor involved in cell homeostasis by regulating lipid asymmetry.

Transbilayer distribution of lipids at nano scale.

There is a limited number of methods to examine transbilayer lipid distribution in biomembranes. We employed freeze-fracture replica-labelling immunoelectron microscopy in combination with lipid-binding proteins and a peptide to examine both transbilayer distribution and lateral distribution of various phospholipids in mammalian cells. Our results indicate that phospholipids are exclusively distributed either in the outer or inner leaflet of human red blood cell (RBC) membranes. In contrast, in nucleated cells, such as human skin fibroblasts and neutrophils, sphingomyelin was distributed in both leaflets while exhibiting characteristic lipid domains in the inner leaflet. Similar to RBCs, lipid asymmetry was maintained both in resting and thrombin-activated platelets. However, the microparticles released from thrombin-activated platelets lost membrane asymmetry. Our results suggest that the microparticles were shed from platelet plasma membrane domains enriched with phosphatidylserine and/or phosphatidylinositol at the outer leaflet. These findings underscore the strict regulation and cell-type specificity of lipid asymmetry in the plasma membrane.

Inner workings and biological impact of phospholipid flippases.

The plasma membrane, trans-Golgi network and endosomal system of eukaryotic cells are populated with flippases that hydrolyze ATP to help establish asymmetric phospholipid distributions across the bilayer. Upholding phospholipid asymmetry is vital to a host of cellular processes, including membrane homeostasis, vesicle biogenesis, cell signaling, morphogenesis and migration. Consequently, defining the identity of flippases and their biological impact has been the subject of intense investigations. Recent work has revealed a remarkable degree of kinship between flippases and cation pumps. In this Commentary, we review emerging insights into how flippases work, how their activity is controlled according to cellular demands, and how disrupting flippase activity causes system failure of membrane function, culminating in membrane trafficking defects, aberrant signaling and disease.

The C-terminal Cytosolic Region of Rim21 Senses Alterations in Plasma Membrane Lipid Composition: INSIGHTS INTO SENSING MECHANISMS FOR PLASMA MEMBRANE LIPID ASYMMETRY.

Yeast responds to alterations in plasma membrane lipid asymmetry and external alkalization via the sensor protein Rim21 in the Rim101 pathway. However, the sensing mechanism used by Rim21 remains unclear. Here, we found that the C-terminal cytosolic domain of Rim21 (Rim21C) fused with GFP was associated with the plasma membrane under normal conditions but dissociated upon alterations in lipid asymmetry or external alkalization. This indicates that Rim21C contains a sensor motif. Rim21C contains multiple clusters of charged residues. Among them, three consecutive Glu residues (EEE motif) were essential for Rim21 function and dissociation of Rim21C from the plasma membrane in response to changes in lipid asymmetry. In contrast, positively charged residues adjacent to the EEE motif were required for Rim21C to associate with the membrane. We therefore propose an "antenna hypothesis," in which Rim21C moves to or from the plasma membrane and functions as the sensing mechanism of Rim21.