18 research outputs found

    CHIP as a membrane-shuttling proteostasis sensor

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    Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function)

    The molecular recognition of phosphatidic acid by an amphipathic helix in Opi1

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    A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important lipid intermediate and signaling lipid at the branch point of these pathways and constantly monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. Here, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for the selective binding to membranes containing PA over phosphatidylserine (PS). The insertion of the AH into the hydrophobic core of the membrane renders Opi1 sensitive to the lipid acyl chain composition as an important factor contributing to the regulation of membrane biogenesis. Based on these findings, we rationally designed the membrane binding properties of Opi1 to control its responsiveness in the physiological context. Using extensive molecular dynamics (MD) simulations, we identified two PA-selective three-finger grips that tightly bind the phosphate headgroup, while interacting less intimately and more transiently with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors

    The molecular recognition of phosphatidic acid by an amphipathic helix in Opi1

    No full text
    A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important intermediate at the branch point of these pathways and is continuously monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. In this study, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for selective binding of PA over phosphatidylserine (PS). The insertion of the AH into the membrane core renders Opi1 sensitive to the lipid acyl chain composition and provides a means to adjust membrane biogenesis. By rational design of the AH, we tune the membrane-binding properties of Opi1 and control its responsiveness in vivo. Using extensive molecular dynamics simulations, we identify two PA-selective three-finger grips that tightly bind the PA phosphate headgroup while interacting less intimately with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors

    Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

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    Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.

    Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment

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    In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach. Keywords: Parkinson’s disease; synucleinopathy; alpha-synuclein; stearoyl-CoA-desaturase; unsaturated fatty acid; oleic acid; lipid droplets; diglyceride; triglyceride; tetramer; inclusion

    Coordination of Storage Lipid Synthesis and Membrane Biogenesis: EVIDENCE FOR CROSS-TALK BETWEEN TRIACYLGLYCEROL METABOLISM AND PHOSPHATIDYLINOSITOL SYNTHESIS*

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    Despite the importance of triacylglycerols (TAG) and steryl esters (SE) in phospholipid synthesis in cells transitioning from stationary-phase into active growth, there is no direct evidence for their requirement in synthesis of phosphatidylinositol (PI) or other membrane phospholipids in logarithmically growing yeast cells. We report that the dga1Δlro1Δare1Δare2Δ strain, which lacks the ability to synthesize both TAG and SE, is not able to sustain normal growth in the absence of inositol (Ino− phenotype) at 37 °C especially when choline is present. Unlike many other strains exhibiting an Ino− phenotype, the dga1Δlro1Δare1Δare2Δ strain does not display a defect in INO1 expression. However, the mutant exhibits slow recovery of PI content compared with wild type cells upon reintroduction of inositol into logarithmically growing cultures. The tgl3Δtgl4Δtgl5Δ strain, which is able to synthesize TAG but unable to mobilize it, also exhibits attenuated PI formation under these conditions. However, unlike dga1Δlro1Δare1Δare2Δ, the tgl3Δtgl4Δtgl5Δ strain does not display an Ino− phenotype, indicating that failure to mobilize TAG is not fully responsible for the growth defect of the dga1Δlro1Δare1Δare2Δ strain in the absence of inositol. Moreover, synthesis of phospholipids, especially PI, is dramatically reduced in the dga1Δlro1Δare1Δare2Δ strain even when it is grown continuously in the presence of inositol. The mutant also utilizes a greater proportion of newly synthesized PI than wild type for the synthesis of inositol-containing sphingolipids, especially in the absence of inositol. Thus, we conclude that storage lipid synthesis actively influences membrane phospholipid metabolism in logarithmically growing cells
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