Antonio Daniel Barbosa

Lipid droplet–organelle interactions: emerging roles in lipid metabolism

Cellular homeostasis depends on the precisely coordinated use of lipids as fuels for energy production, building blocks for membrane biogenesis or chemical signals for intra-cellular and inter-cellular communication. Lipid droplets (LDs) are universally conserved dynamic organelles that can store and mobilize fatty acids and other lipid species for their multiple cellular roles. Increasing evidence suggests that contact zones between LDs and other organelles play important roles in the trafficking of lipids and in the regulation of lipid metabolism. Here we review recent advances regarding the nature and functional relevance of interactions between LDs and other organelles-particularly the endoplasmic reticulum (ER), LDs, mitochondria and vacuoles-that highlight their importance for lipid metabolism.

Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p

Binding and dephosphorylation of the yeast lipin Pah1p by its phosphatase Nem1p-Spo7p is essential for its membrane targeting and is mediated by a C-terminal acidic stretch on Pah1p. This results in the recruitment of Pah1p to the vicinity of lipid droplets, where it controls triglyceride and droplet biogenesis in an acidic tail–dependent manner.

Lipid partitioning at the nuclear envelope controls membrane biogenesis

Cells adjust the flux of lipid intermediates toward membranes or storage in response to their metabolic status. In response to growth cues, spatiotemporal activation of Pah1 at discrete subdomains of the nuclear envelope acts as a switch to promote lipid storage. This lipid rewiring controls organelle morphology.

Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1-3.

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs in Saccharomyces cerevisiae, demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targeting in vivo and in vitro. Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.

Function of lipid droplet-organelle interactions in lipid homeostasis

Storage of non-polar lipids in ubiquitous eukaryotic organelles, lipid droplets (LDs), prevents the toxic consequences of unesterified fatty acids and provides a lipid reservoir that can be promptly used to satisfy cellular needs under multiple metabolic and physiological conditions. Tight temporal and spatial control of LD biogenesis and mobilization of neutral lipids is essential for the correct channelling of lipid intermediates to their various cellular destinations and the maintenance of cellular homeostasis. These functions are mediated by multiple interactions between LDs and other intracellular organelles that are required for the delivery of stored lipids. Here we review recent advances in the interactions of LDs with the endoplasmic reticulum (ER), mitochondria and vacuole/lysosome. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae

Background: Diacylglycerol kinase produces phosphatidate, a major precursor for the synthesis of membrane phospholipids. Results: The expression of diacylglycerol kinase is induced by the Reb1p transcription factor, and the resulting activity increase is essential for the enzyme function in phospholipid synthesis. Conclusion: The Reb1p-mediated transcriptional activation regulates the expression of diacylglycerol kinase activity. Significance: Diacylglycerol kinase is regulated at the level of transcription. In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, −166 to −160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in β-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism.

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress

Summary Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.

Spatial distribution of lipid droplets during starvation: Implications for lipophagy.

ABSTRACT Survival during starvation depends largely on metabolic energy, which is stored in the form of neutral lipids in specialized organelles known as lipid droplets. The precursors for the synthesis of neutral lipids are also used for membrane biogenesis, which is required for cell growth and proliferation. Therefore cells must possess mechanisms to preferentially channel lipid precursors toward either membrane synthesis or lipid droplet storage, in response to nutrient status. How this partitioning is spatially regulated within the endoplasmic reticulum (ER) where lipid droplets co-localize, remains poorly understood. We have recently shown that at the onset of starvation lipid droplets concentrate at a perinuclear ER subdomain flanking the nucleus-vacuole junction (NVJ) and that this is crucial for maintaining proper nuclear shape and ER membrane organization. Here we show that disruption of the NVJ does not block the translocation and internalization of lipid droplets into the vacuole for their degradation, which takes place at later stages of starvation. We propose that alternative pathways of lipid droplet translocation from the ER to the vacuole may exist to enable stationary phase-induced lipophagy.

The professional knowledge base of science teaching

The scientific bases considered necessary for science teaching have been changing during recent decades owing to the evolution of curriculum planning and trends in science education. In this book, ...