Organelle Compartments Research Articles

Metabolic Engineering of Saccharomyces cerevisiae for Production of Canthaxanthin, Zeaxanthin, and Astaxanthin.

The sustainable production of natural compounds is increasingly important in today's industrial landscape. This study investigates the metabolic engineering of Saccharomyces cerevisiae for the efficient biosynthesis of valuable carotenoids: canthaxanthin, zeaxanthin, and astaxanthin. Utilizing a tailored parental yeast strain, Sp_Bc, we optimized the carotenoid pathway by screening and identifying CrtW and CrtZ enzymatic variants. The CrtW variant from Bradyrhizobium sp. achieved a canthaxanthin titer of 425.1 ± 69.1 µg/L, while the CrtZ variant from Pantoea ananatis achieved a zeaxanthin titer of 70.5 ± 10.8 µg/L. Additionally, we optimized carotenoid production by exploring enzyme fusion strategies for all three studied carotenoids and organelle compartmentalization specifically for enhancing astaxanthin synthesis. We further improved carotenoid production by integrating the optimal gene constructs into the yeast genome and deleting the GAL80 gene, enabling the use of sucrose as a carbon source. The engineered strain Sp_Bc-Can001 ∆gal80 was evaluated in a 5 L bioreactor fermentation, achieving a notable canthaxanthin titer of 60.36 ± 1.51 mg/L using sucrose. This research conclusively establishes S. cerevisiae as a viable platform for efficient carotenoid biosynthesis and, for the first time in this yeast system, illustrates sucrose's viability as a carbon source for canthaxanthin production. These findings pave the way for sustainable, cost-effective carotenoid production at an industrial scale.

Emergent properties of melanin-inspired peptide/RNA condensates.

Most biocatalytic processes in eukaryotic cells are regulated by subcellular microenvironments such as membrane-bound or membraneless organelles. These natural compartmentalization systems have inspired the design of synthetic compartments composed of a variety of building blocks. Recently, the emerging field of liquid-liquid phase separation has facilitated the design of biomolecular condensates composed of proteins and nucleic acids, with controllable properties including polarity, diffusivity, surface tension, and encapsulation efficiency. However, utilizing phase-separated condensates as optical sensors has not yet been attempted. Here, we were inspired by the biosynthesis of melanin pigments, a key biocatalytic process that is regulated by compartmentalization in organelles, to design minimalistic biomolecular condensates with emergent optical properties. Melanins are ubiquitous pigment materials with a range of functionalities including photoprotection, coloration, and free radical scavenging activity. Their biosynthesis in the confined melanosomes involves oxidation-polymerization of tyrosine (Tyr), catalyzed by the enzyme tyrosinase. We have now developed condensates that are formed by an interaction between a Tyr-containing peptide and RNA and can serve as both microreactors and substrates for tyrosinase. Importantly, partitioning of Tyr into the condensates and subsequent oxidation-polymerization gives rise to unique optical properties including far-red fluorescence. We now demonstrate that individual condensates can serve as sensors to detect tyrosinase activity, with a limit of detection similar to that of synthetic fluorescent probes. This approach opens opportunities to utilize designer biomolecular condensates as diagnostic tools for various disorders involving abnormal enzymatic activity.

Advances in the production of chemicals by organelle compartmentalization in Saccharomyces cerevisiae

As a generally-recognized-as-safe microorganism, Saccharomyces cerevisiae is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in S. cerevisiae by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, S. cerevisiae has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in S. cerevisiae are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.

Compartmentalization engineering of yeasts to overcome precursor limitations and cytotoxicity in terpenoid production.

Metabolic engineering strategies for terpenoid production have mainly focused on bottlenecks in the supply of precursor molecules and cytotoxicity to terpenoids. In recent years, the strategies involving compartmentalization in eukaryotic cells has rapidly developed and have provided several advantages in the supply of precursors, cofactors and a suitable physiochemical environment for product storage. In this review, we provide a comprehensive analysis of organelle compartmentalization for terpenoid production, which can guide the rewiring of subcellular metabolism to make full use of precursors, reduce metabolite toxicity, as well as provide suitable storage capacity and environment. Additionally, the strategies that can enhance the efficiency of a relocated pathway by increasing the number and size of organelles, expanding the cell membrane and targeting metabolic pathways in several organelles are also discussed. Finally, the challenges and future perspectives of this approach for the terpenoid biosynthesis are also discussed.

Organelle-Selective Membrane Labeling through Phospholipase D-Mediated Transphosphatidylation.

The specialized functions of eukaryotic organelles have motivated chemical approaches for their selective tagging and visualization. Here, we develop chemoenzymatic tools using metabolic labeling of abundant membrane lipids for selective visualization of organelle compartments. Synthetic choline analogues with three N-methyl substituents replaced with 2-azidoethyl and additional alkyl groups enabled the generation of corresponding derivatives of phosphatidylcholine (PC), a ubiquitous and abundant membrane phospholipid. Subsequent bioorthogonal tagging via the strain-promoted azide-alkyne cycloaddition (SPAAC) with a single cyclooctyne-fluorophore reagent enabled differential labeling of the endoplasmic reticulum, the Golgi complex, mitochondria, and lysosomes depending upon the substitution pattern at the choline ammonium center. Key to the success of this strategy was the harnessing of both the organic cation transporter OCT1 to enable cytosolic delivery of these cationic metabolic probes and endogenous phospholipase D enzymes for rapid, one-step metabolic conversion of the choline analogues to the desired lipid products. Detailed analysis of the trafficking kinetics of both the SPAAC-tagged fluorescent PC analogues and their non-fluorescent, azide-containing precursors revealed that the latter exhibit time-dependent differences in organelle selectivity, suggesting their use as probes for visualizing intracellular lipid transport pathways. By contrast, the stable localizations of the fluorescent PC analogues will allow applications not only for organelle-selective imaging but also for local modulation of physiological events with organelle-level precision by tethering of bioactive small molecules, via click chemistry, within defined subcellular membrane environments.

Imaging and Editing the Phospholipidome.

Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component of membranes. They make up the majority of the lipid bilayer and play both structural and functional roles. Each organelle has a different phospholipid composition critical for its function that results from dynamic interplay and regulation of numerous lipid-metabolizing enzymes and lipid transporters. Because lipid structures and localizations are not directly genetically encoded, chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and in general decoding the functions of lipids in cells.In this Account, we provide an overview of our recent efforts in this space focused on two overarching and complementary goals: imaging and editing the phospholipidome. On the imaging front, we have harnessed the power of bioorthogonal chemistry to develop fluorescent reporters of specific lipid pathways. Substantial efforts have centered on phospholipase D (PLD) signaling, which generates the humble lipid phosphatidic acid (PA) that acts variably as a biosynthetic intermediate and signaling agent. Though PLD is a hydrolase that generates PA from abundant phosphatidylcholine (PC) lipids, we have exploited its transphosphatidylation activity with exogenous clickable alcohols followed by bioorthogonal tagging to generate fluorescent lipid reporters of PLD signaling in a set of methods termed IMPACT.IMPACT and its variants have facilitated many biological discoveries. Using the rapid and fluorogenic tetrazine ligation, it has revealed the spatiotemporal dynamics of disease-relevant G protein-coupled receptor signaling and interorganelle lipid transport. IMPACT using diazirine photo-cross-linkers has enabled identification of lipid-protein interactions relevant to alcohol-related diseases. Varying the alcohol reporter can allow for organelle-selective labeling, and varying the bioorthogonal detection reagent can afford super-resolution lipid imaging via expansion microscopy. Combination of IMPACT with genome-wide CRISPR screening has revealed genes that regulate physiological PLD signaling.PLD enzymes themselves can also act as tools for precision editing of the phospholipid content of membranes. An optogenetic PLD for conditional blue-light-stimulated synthesis of PA on defined organelle compartments led to the discovery of the role of organelle-specific pools of PA in regulating oncogenic Hippo signaling. Directed enzyme evolution of PLD, enabled by IMPACT, has yielded highly active superPLDs with broad substrate tolerance and an ability to edit membrane phospholipid content and synthesize designer phospholipids in vitro. Finally, azobenzene-containing PA analogues represent an alternative, all-chemical strategy for light-mediated control of PA signaling.Collectively, the strategies described here summarize our progress to date in tackling the challenge of assigning precise functions to defined pools of phospholipids in cells. They also point to new challenges and directions for future study, including extension of imaging and membrane editing tools to other classes of lipids. We envision that continued application of bioorthogonal chemistry, optogenetics, and directed evolution will yield new tools and discoveries to interrogate the phospholipidome and reveal new mechanisms regulating phospholipid homeostasis and roles for phospholipids in cell signaling.

Targeting Apollo-NADP+ to Image NADPH Generation in Pancreatic Beta-Cell Organelles.

NADPH/NADP+ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP+. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP+ sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP+ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single- and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP+ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

Phase Separation in the Nucleus and at the Nuclear Periphery during Post-Mitotic Nuclear Envelope Reformation.

Membrane-enclosed organelle compartmentalization is not the only way by which cell processes are spatially organized. Phase separation is emerging as a new driver in the organization of membrane-less compartments and biological processes. Liquid–liquid phase separation has been indicated as a new way to control the kinetics of molecular reactions and is based on weak multivalent interactions affecting the stoichiometry of the molecules involved. In the nucleus, liquid–liquid phase separation may represent an ancestral means of controlling genomic activity by forming discrete chromatin regions, regulating transcriptional activity, contributing to the assembly of DNA damage response foci, and controlling the organization of chromosomes. Liquid–liquid phase separation also contributes to chromatin function through its role in the reorganization of the nuclear periphery in the post-mitotic phase. Herein, we describe the basic principles regulating liquid–liquid phase separation, analyze examples of phase separation occurring in the nucleus, and dedicate attention to the implication of liquid–liquid phase separation in the reorganization of the nuclear periphery by the endosomal sorting complexes required for transport (ESCRT) machinery. Although some caution is warranted, current scientific knowledge allows for the hypothesis that many factors and processes in the cell are yet to be discovered which are functionally associated with phase separation.

Acyl-Coa Thioesterases: A Rheostat That Controls Activated Fatty Acids Modulates Dengue Virus Serotype 2 Replication.

During infection with dengue viruses (DENVs), the lipid landscape within host cells is significantly altered to assemble membrane platforms that support viral replication and particle assembly. Fatty acyl-CoAs are key intermediates in the biosynthesis of complex lipids that form these membranes. They also function as key signaling lipids in the cell. Here, we carried out loss of function studies on acyl-CoA thioesterases (ACOTs), a family of enzymes that hydrolyze fatty acyl-CoAs to free fatty acids and coenzyme A, to understand their influence on the lifecycle of DENVs. The loss of function of the type I ACOTs 1 (cytoplasmic) and 2 (mitochondrial) together significantly increased DENV serotype 2 (DENV2) viral replication and infectious particle release. However, isolated knockdown of mitochondrial ACOT2 significantly decreased DENV2 protein translation, genome replication, and infectious virus release. Furthermore, loss of ACOT7 function, a mitochondrial type II ACOT, similarly suppressed DENV2. As ACOT1 and ACOT2 are splice variants, these data suggest that functional differences and substrate specificities due to the location (cytosol and mitochondria, respectively) of these proteins may account for the differences in DENV2 infection phenotype. Additionally, loss of mitochondrial ACOT2 and ACOT7 expression also altered the expression of several ACOTs located in multiple organelle compartments within the cell, highlighting a complex relationship between ACOTs in the DENV2 virus lifecycle.

SARS-CoV-2 infection enhances mitochondrial PTP complex activity to perturb cardiac energetics

SummarySARS-CoV-2 is a newly identified coronavirus that causes the respiratory disease called coronavirus disease 2019 (COVID-19). With an urgent need for therapeutics, we lack a full understanding of the molecular basis of SARS-CoV-2-induced cellular damage and disease progression. Here, we conducted transcriptomic analysis of human PBMCs, identified significant changes in mitochondrial, ion channel, and protein quality-control gene products. SARS-CoV-2 proteins selectively target cellular organelle compartments, including the endoplasmic reticulum and mitochondria. M-protein, NSP6, ORF3A, ORF9C, and ORF10 bind to mitochondrial PTP complex components cyclophilin D, SPG-7, ANT, ATP synthase, and a previously undescribed CCDC58 (coiled-coil domain containing protein 58). Knockdown of CCDC58 or mPTP blocker cyclosporin A pretreatment enhances mitochondrial Ca2+ retention capacity and bioenergetics. SARS-CoV-2 infection exacerbates cardiomyocyte autophagy and promotes cell death that was suppressed by cyclosporin A treatment. Our findings reveal that SARS-CoV-2 viral proteins suppress cardiomyocyte mitochondrial function that disrupts cardiomyocyte Ca2+ cycling and cell viability.

Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica

Metabolic engineering approaches for the production of high-value chemicals in microorganisms mostly use the cytosol as general reaction vessel. However, sequestration of enzymes and substrates, and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Organelle compartmentalization in eukaryotic cells suggests ways for overcoming these challenges. Here we have explored this strategy by expressing the astaxanthin biosynthesis pathway in sub-organelles of the oleaginous yeast Yarrowia lipolytica. We first showed that fusion of the two enzymes converting β-carotene to astaxanthin, β-carotene ketolase and hydroxylase, performs better than the expression of individual enzymes. We next evaluated the pathway when expressed in compartments of lipid body, endoplasmic reticulum or peroxisome, individually and in combination. Targeting the astaxanthin pathway to subcellular organelles not only accelerated the conversion of β-carotene to astaxanthin, but also significantly decreased accumulation of the ketocarotenoid intermediates. Anchoring enzymes simultaneously to all three organelles yielded the largest increase of astaxanthin synthesis, and ultimately produced 858 mg/L of astaxanthin in fed-batch fermentation (a 141-fold improvement over the initial strain). Our study is expected to help unlock the full potential of subcellular compartments and advance LB-based compartmentalized isoprenoid biosynthesis in Y. lipolytica.

LRRK2 recruitment, activity, and function in organelles.

Protein coding mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD), and noncoding variations around the gene increase the risk of developing sporadic PD. It is generally accepted that pathogenic LRRK2 mutations increase LRRK2 kinase activity, resulting in a toxic hyperactive protein that is inferred to lead to the PD phenotype. LRRK2 has long been linked to different membrane trafficking events, but the specific role of LRRK2 in these events has been difficult to resolve. Recently, several papers have reported the activation and translocation of LRRK2 to cellular organelles under specific conditions, which suggests that LRRK2 may influence intracellular membrane trafficking. Here, we review what is known about the role of LRRK2 at various organelle compartments.

High-efficiency plastome base editing in rice with TAL cytosine deaminase

High-efficiency plastome base editing in rice with TAL cytosine deaminase

Manipulation of Host Cell Organelles by Intracellular Pathogens.

Pathogenic intracellular bacteria, parasites and viruses have evolved sophisticated mechanisms to manipulate mammalian host cells to serve as niches for persistence and proliferation. The intracellular lifestyles of pathogens involve the manipulation of membrane-bound organellar compartments of host cells. In this review, we described how normal structural organization and cellular functions of endosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, or lipid droplets are targeted by microbial virulence mechanisms. We focus on the specific interactions of Salmonella, Legionella pneumophila, Rickettsia rickettsii, Chlamydia spp. and Mycobacterium tuberculosis representing intracellular bacterial pathogens, and of Plasmodium spp. and Toxoplasma gondii representing intracellular parasites. The replication strategies of various viruses, i.e., Influenza A virus, Poliovirus, Brome mosaic virus, Epstein-Barr Virus, Hepatitis C virus, severe acute respiratory syndrome virus (SARS), Dengue virus, Zika virus, and others are presented with focus on the specific manipulation of the organelle compartments. We compare the specific features of intracellular lifestyle and replication cycles, and highlight the communalities in mechanisms of manipulation deployed.

Abstract 2411: Cancer cell lines meta-analysis according to both short-term and long-term responses to Tumor Treating Fields (TTFields)

Abstract Tumor Treating Fields (TTFields) therapy is an approved modality for the treatment of glioblastoma. These alternating electric fields were shown to exert an inhibitory effect in numerous cancer cell lines with some variability in the response of different cell lines. The goal of the present study is to compare characteristics of cell lines based on their response pattern to TTFields. Forty one different human cancerous cell lines were treated for 72 hours with TTFields at their optimal frequency with the same nominal intensity (1.7 V/cm). Two response values were quantified. The first cytotoxic response, is considered a short-term quantification of the response. The second clonogenic response, is considered a long-term response to TTFields. Genomic analysis of 26 cell lines was performed based on the Cancer Cell Line Encyclopedia (CCLE) database. Association of mutated genes, mutations and copy number variations with cell lines response to TTFields was investigated. TTFields application demonstrated varying degree of cytotoxic effect in all cell lines tested. The inhibitory response to TTFields was found to be distributed around an average of 50% with a cytotoxic effects ranging between 14% and 86% reductions in cell counts, and a clonogenic effect ranging between no effect and 88% reduction in the number of colonies. The “Overall response” was considered as the multiplication of the two values and represents the response both in short-term and long-term effects of TTFields. Lower values of all response quantifications indicate a better response to TTFields. Cell lines were partitioned to responding and non-responding cell lines according to their “Overall response”. Response to treatment doesn't seem to be affected by the number of mutations in each of the cell lines, and not by the type of the cancer the cell lines originated from. Mutations in genes that associate with the response to TTFields are enriched in GO terms that include muscle function and the intracellular organelle compartment. Mutation association with response to TTFields revealed mutations in genes related to the cytoskeleton, mitotic spindle, receptors and membrane transporters. This multi parameter, large scale comparison of cancerous cell line response to TTFields demonstrate the broad effectiveness of TTFields in various cell lines and define the optimal frequency to be applied for each cell line. The data presented in this work, suggest that beside their anti-mitotic properties, TTFields may have effects on other cellular pathways. Citation Format: Kerem Wainer Katsir, Gitit Lavy Shahaf, Moshe Giladi, Rosa S. Schneiderman, Noa Urman, Karnit Gotlieb, Einav Zeevi, Yaara Porat, Mijal Munster, Adrian Kinzel, Uri Weinberg, Eilon D. Kirson, Yoram Palti. Cancer cell lines meta-analysis according to both short-term and long-term responses to Tumor Treating Fields (TTFields) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2411.

Biomimetic Models to Investigate Membrane Biophysics Affecting Lipid-Protein Interaction.

Biological membranes are highly dynamic in their ability to orchestrate vital mechanisms including cellular protection, organelle compartmentalization, cellular biomechanics, nutrient transport, molecular/enzymatic recognition, and membrane fusion. Controlling lipid composition of different membranes allows cells to regulate their membrane characteristics, thus modifying their physical properties to permit specific protein interactions and drive structural function (membrane deformation facilitates vesicle budding and fusion) and signal transduction. Yet, how lipids control protein structure and function is still poorly understood and needs systematic investigation. In this review, we explore different in vitro membrane models and summarize our current understanding of the interplay between membrane biophysical properties and lipid–protein interaction, taken as example few proteins involved in muscular activity (dystrophin), digestion and Legionella pneumophila effector protein DrrA. The monolayer model with its movable barriers aims to mimic any membrane deformation while surface pressure modulation imitates lipid packing and membrane curvature changes. It is frequently used to investigate peripheral protein binding to the lipid headgroups. Examples of how lipid lateral pressure modifies protein interaction and organization within the membrane are presented using various biophysical techniques. Interestingly, the shear elasticity and surface viscosity of the monolayer will increase upon specific protein(s) binding, supporting the importance of such mechanical link for membrane stability. The lipid bilayer models such as vesicles are not only used to investigate direct protein binding based on the lipid nature, but more importantly to assess how local membrane curvature (vesicles with different size) influence the binding properties of a protein. Also, supported lipid bilayer model has been used widely to characterize diffusion law of lipids within the bilayer and/or protein/biomolecule binding and diffusion on the membrane. These membrane models continue to elucidate important advances regarding the dynamic properties harmonizing lipid–protein interaction.

Isoform‐resolved correlation analysis between mRNA abundance regulation and protein level degradation

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post‐translational turnover, we devised a strategy combining pulse stable isotope‐labeled amino acids in cells (pSILAC), data‐independent acquisition mass spectrometry (DIA‐MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome‐wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.

Curved Lipid Interfaces Studied with Grazing Incident SANS

Curved cell membranes are important for the function of the cell, both for compartmentalization in organelles within the cell, as well as for cellular mitosis. It has also been shown that the curvature of a lipid membrane can affect the concentration of membrane bound proteins. In this project we have used semiconductor nanowires in order to study the effect of curvature on phospholipid bilayers and membrane proteins. We have previously demonstrated the formation of a supported phospholipid bilayer on nano wires via vesicle fusion, which can be used as a proxy to curved membranes. Based on fluorescence recovery after photobleaching, FRAP, measurement, the phospholipid bilayers were found to follow the contours of the nano wires as continuous and fluid, locally curved bilayers. However these data do not reveal the coverage, composition and the dimensions of the bilayer. In this project we aim to revel this using grazing incident small angle neutron scattering (GISANS). For this purpose we have performed GISANS experiments at the KSW-1 at FRM II (Germany), VSANS at NIST (USA) and SANS2D at ISIS (UK) with phospholipid covered nanowires. The nanowires were covered with a phospholipid bilayer obtained from deposition from vesicular dispersion of mixture of 20mol% DOPE in DOPC. In these experiments we have successfully shown the formation of a lipid bilayer and the subsequent binding of membrane protein. These results shows that nanowire supported lipid bilayer is an excellent way to study biding membrane protein to curved lipid interfaces.

APEX2-mediated proximity labeling resolves protein networks in Saccharomycescerevisiae cells.

Enzyme-catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin-phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane-enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast.