Metabolic control of cytosolic-facing pools of diacylglycerol in budding yeast.

Diacylglycerol (DAG) is a key signaling lipid and intermediate in lipid metabolism. Our knowledge of DAG distribution and dynamics in cell membranes is limited. Using live-cell fluorescence microscopy we investigated the localization of yeast cytosolic-facing pools of DAG in response to conditions where lipid homeostasis and DAG levels were known to be altered. Two main pools were monitored over time using DAG sensors. One pool was associated with vacuolar membranes and the other localized to sites of polarized growth. Dynamic changes in DAG distribution were observed during resumption of growth from stationary phase, when DAG is used to support phospholipid synthesis for membrane proliferation. Vacuolar membranes experienced constant morphological changes displaying DAG enriched microdomains coexisting with liquid-disordered areas demarcated by Vph1. Formation of these domains was dependent on triacylglycerol (TAG) lipolysis. DAG domains and puncta were closely connected to lipid droplets. Lack of conversion of DAG to phosphatidate in growth conditions dependent on TAG mobilization, led to the accumulation of DAG in a vacuolar-associated compartment, impacting the polarized distribution of DAG at budding sites. DAG polarization was also regulated by phosphatidylserine synthesis/traffic and sphingolipid synthesis in the Golgi.

Lipids and Membrane Microdomains: The Glycerolipid and Alkylphosphocholine Class of Cancer Chemotherapeutic Drugs.

Synthetic antitumor lipids are metabolically stable lysophosphatidylcholine derivatives, encompassing a class of non-mutagenic drugs that selectively target cancerous cells. In this chapter we review the literature as relates to the clinical efficacy of these antitumor lipid drugs and how our understanding of their mode of action has evolved alongside key advances in our knowledge of membrane structure, organization, and function. First, the history of the development of this class of drugs is described, providing a summary of clinical outcomes of key members including edelfosine, miltefosine, perifosine, erufosine, and erucylphosphocholine. A detailed description of the biophysical properties of these drugs and specific drug-lipid interactions which may contribute to the selectivity of the antitumor lipids for cancer cells follows. An updated model on the mode of action of these lipid drugs as membrane disorganizing agents is presented. Membrane domain organization as opposed to targeting specific proteins on membranes is discussed. By altering membranes, these antitumor lipids inhibit many survival pathways while activating pro-apoptotic signals leading to cell demise.

Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution.

Actively maintained close appositions between organelle membranes, also known as contact sites, enable the efficient transfer of biomolecules between cellular compartments. Several such sites have been described as well as their tethering machineries. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize contact site proteomes, we established a high-throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, several of which are poorly characterized, on the background of 1165 strains expressing a mCherry-tagged yeast protein that has a cellular punctate distribution (a hallmark of contact sites), under regulation of the strong TEF2 promoter. By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified three homologs of Vps13 and Atg2 that are residents of multiple contact sites. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell. Overall, our analysis expands the universe of contact site residents and effectors and creates a rich database to mine for new functions, tethers, and regulators.

Tubular ER Associates With Diacylglycerol-Rich Structures During Lipid Droplet Consumption.

Growth resumption from stationary phase in Saccharomyces cerevisiae, is characterized by lipid droplet (LD) consumption and channeling of lipid precursors toward synthesis of membranes. We have previously determined that triacylglycerol lipolysis contributes to a pool of diacylglycerol (DAG) associated with the yeast vacuole that is enriched in structures that are in close proximity to LDs. In this study we have monitored these structures using a DAG sensor fused to GFP during isolation of LDs. A unique fraction containing the DAG sensor, with low presence of LDs, was identified. Membranes enriched in the DAG probe were obtained by immunoaffinity purification using a GFP nanobody, and the associated proteome was investigated by mass spectrometry. It was determined this LD-associated fraction was enriched in proteins known to shape the tubular endoplasmic reticulum (ER) like Yop1, Sey1, Rtn1, and Rtn2. Consistently, cells lacking three of these proteins (rtn1Δ rtn2Δ yop1Δ) exhibited delayed LD consumption, larger LDs and abnormal LD distribution. In addition, the triple mutant displayed aberrant localization of the DAG sensor after 5 h of growth resumption from stationary phase. Manipulation of DAG levels by overexpression of the DAG kinase Dgk1, impacted localization of the DAG probe and affected fitness of the triple mutant. Altogether these results link LD consumption to tubular ER expansion as a gateway of lipid precursors that otherwise accumulate in vacuolar associated membranes or other internal compartments. Furthermore, conversion of DAG to phosphatidic acid (PA) in the absence of a functional tubular ER was toxic to cells, suggesting the ratio of PA to DAG is critical to allow growth progression.

pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels.

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3)P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes.

A Signaling Lipid Associated with Alzheimer's Disease Promotes Mitochondrial Dysfunction.

Fundamental changes in the composition and distribution of lipids within the brain are believed to contribute to the cognitive decline associated with Alzheimer's disease (AD). The mechanisms by which these changes in lipid composition affect cellular function and ultimately cognition are not well understood. Although "candidate gene" approaches can provide insight into the effects of dysregulated lipid metabolism they require a preexisting understanding of the molecular targets of individual lipid species. In this report we combine unbiased gene expression profiling with a genome-wide chemogenomic screen to identify the mitochondria as an important downstream target of PC(O-16:0/2:0), a neurotoxic lipid species elevated in AD. Further examination revealed that PC(O-16:0/2:0) similarly promotes a global increase in ceramide accumulation in human neurons which was associated with mitochondrial-derived reactive oxygen species (ROS) and toxicity. These findings suggest that PC(O-16:0/2:0)-dependent mitochondrial dysfunction may be an underlying contributing factor to the ROS production associated with AD.

Tracking Diacylglycerol and Phosphatidic Acid Pools in Budding Yeast.

Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed.

Disruption of lipid domain organization in monolayers of complex yeast lipid extracts induced by the lysophosphatidylcholine analogue edelfosine in vivo.

The lysophosphatidylcholine analogue edelfosine is a potent antitumor and antiparasitic drug that targets cell membranes. Previous studies have shown that edelfosine alters membrane domain organization inducing internalization of sterols and endocytosis of plasma membrane transporters. These early events affect signaling pathways that result in cell death. It has been shown that edelfosine preferentially partitions into more rigid lipid domains in mammalian as well as in yeast cells. In this work we aimed at investigating the effect of edelfosine on membrane domain organization using monolayers prepared from whole cell lipid extracts of cells treated with edelfosine compared to control conditions. In Langmuir monolayers we were able to detect important differences to the lipid packing of the membrane monofilm. Domain formation visualized by means of Brewster angle microscopy also showed major morphological changes between edelfosine treated versus control samples. Importantly, edelfosine resistant cells defective in drug uptake did not display the same differences. In addition, co-spread samples of control lipid extracts with edelfosine added post extraction did not fully mimic the results obtained with lipid extracts from treated cells. Altogether these results indicate that edelfosine induces changes in membrane domain organization and that these changes depend on drug uptake. Our work also validates the use of monolayers derived from complex cell lipid extracts combined with Brewster angle microscopy, as a sensitive approach to distinguish between conditions associated with susceptibility or resistance to lysophosphatidylcholine analogues.