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UBXD8 lipidomics from whole cells (Part 1)

ABSTRACT: The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipidome of cells. LC-MS/MS lipidomics found significant changes in distinct lipid species in UBXD8 knockout cells, in particular in saturated or mono-unsaturated lipid species. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark in a variety of human disorders such as neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on UBXD8.

ORGANISM(S): Human Homo Sapiens

TISSUE(S): Cultured Cells

SUBMITTER: John Purdy

PROVIDER: ST002421 | MetabolomicsWorkbench | Fri Dec 30 00:00:00 GMT 2022

REPOSITORIES: MetabolomicsWorkbench

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UBXD8 lipidomics from whole cells (Part 2)

2023-01-01 | ST002422 | MetabolomicsWorkbench

The p97-UBXD8 complex regulates ER-Mitochondria contact sites by altering membrane lipid saturation and composition

Project description:The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we show that the p97 AAA-ATPase and its ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulate the prevalence of ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in saturated or monounsaturated lipid species in UBXD8 knockout cells. We show that loss of p97-UBXD8 results in perturbed contacts due to an increase in membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. These aberrant contacts can be rescued by supplementation with unsaturated fatty acids or overexpression of SCD1. Notably, we find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a p97-UBXD8 dependent manner.

2023-01-25 | PXD039061 | Pride

A new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress

Project description:Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remained insufficiently characterized. Here we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers a dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.

2024-03-05 | PXD048553 | Pride

A fast-acting lipid checkpoint in G1 prevents mitotic defects

Project description:Lipid synthesis must increase during the cell cycle to double membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis in non-transformed cells. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic failure and cell death, which are triggered by low lipid synthesis much later in mitosis.

2024-02-22 | GSE254479 | GEO

Interorganelle lipid flux revealed by enzymatic mass tagging in vivo

Project description:The distinct activities of cellular organelles are dependent on the proper function of their membranes. Coordinated membrane biogenesis of the different organelles necessitates interorganelle transport of lipids from their site of synthesis to their destination membranes. Several proteins and trafficking pathways have been proposed to participate in lipid distribution, but despite the basic importance of this process, in vivo evidence linking the absence of putative transport pathways to specific transport defects remains scarce. An obvious reason for this scarcity is the near absence of in vivo lipid trafficking assays. Here we introduce a versatile method named METALIC (Mass tagging-Enabled TrAcking of Lipids In Cells) to track interorganelle lipid flux inside living cells. In this strategy, two enzymes, one directed to a “donor” and the other to an “acceptor” organelle, add two distinct mass tags to lipids. Mass spectrometry-based detection of lipids bearing the two mass tags is then used as a proxy for lipid exchange between the two organelles. By applying this approach to ER and mitochondria, we show that the ERMES and Vps13-Mcp1 complexes have lipid transport activity in vivo, and unravel their relative contributions to ER-mitochondria lipid exchange.

2022-03-22 | MTBLS3415 | MetaboLights

Disturbed intramitochondrial phosphatidic acid transport impairs cellular stress signaling

Project description:Lipid transfer proteins of the Ups1/PRELID1 family facilitate the transport of phospholipids across the intermembrane space of mitochondria in a lipid-specific manner. Heterodimeric complexes of yeast Ups1/Mdm35 or human PRELID1/TRIAP1 shuttle phosphatidic acid (PA) synthesized in the endoplasmic reticulum (ER) to the inner membrane, where it is converted to cardiolipin (CL), the signature phospholipid of mitochondria. Loss of Ups1/PRELID1 proteins impairs the accumulation of CL and broadly affects mitochondrial structure and function. Unexpectedly and unlike yeast cells lacking the cardiolipin synthase Crd1, Ups1 deficient yeast cells exhibit glycolytic growth defects, pointing to functions of Ups1-mediated PA transfer beyond CL synthesis. Here, we show that the disturbed intramitochondrial transport of PA in ups1D cells leads to altered phospholipid composition of the ER membrane, independent of disturbances in CL synthesis. The impaired flux of PA into mitochondria is associated with the increased synthesis of phosphatidylcholine (PC) and a reduced phosphatidylethanolamine (PE)/PC ratio in the ER of ups1D cells which suppresses the unfolded protein response (UPR). Moreover, we observed inhibition of TORC1 signaling in these cells. Activation of either UPR by ER protein stress or of TORC1 signaling by disruption of its negative regulator, the SEACIT complex, increased cytosolic protein synthesis and restored glycolytic growth of ups1D cells. These results demonstrate that PA influx into mitochondria is required to preserve ER membrane homeostasis and that its disturbance is associated with impaired glycolytic growth and cellular stress signaling.

2021-01-21 | GSE160861 | GEO

ACBD5 phosphorylationsite analysis

Project description:Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form membrane contacts, which are mediated by the peroxisomal membrane proteins acyl-coenzyme A-binding domain protein 4 and 5 (ACBD4/5) which bind to the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). ACBD4/5 bind to the major sperm protein (MSP) domain of VAPB via their FFAT-like [two phenylalanines (FF) in an acidic tract] motif. The molecular mechanisms which regulate membrane contact site formation and dynamics are not well explored, in particular in mammalian cells. Here, we reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation.

2022-01-10 | PXD018005 | Pride

Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2

Project description:The regulation of membrane lipid composition is critical for cellular homeostasis. Cells are particularly sensitive to phospholipid saturation, with increased saturation causing membrane rigidification and lipotoxicity. How mammalian cells sense membrane lipid composition and reverse fatty acid (FA)-induced membrane rigidification is poorly understood. Here we systematically identify proteins that differ between mammalian cells fed saturated versus unsaturated FAs. The most differentially expressed proteins were two ER-resident polytopic membrane proteins: the E3 ubiquitin ligase RNF145 and the lipid hydrolase ADIPOR2. In unsaturated lipid membranes, RNF145 is stable, promoting its lipid-sensitive interaction, ubiquitination and degradation of ADIPOR2. When membranes become enriched in saturated FAs, RNF145 is rapidly auto-ubiquitinated and degraded, stabilising ADIPOR2, whose hydrolase activity restores lipid homeostasis and prevents lipotoxicity. We therefore identify RNF145 as a FA-responsive ubiquitin ligase which, together with ADIPOR2, define an autoregulatory pathway that controls cellular membrane lipid homeostasis and prevents acute lipotoxic stress.

2022-08-10 | PXD034835 | Pride

PUB11-dependent ubiquitination of ALA10

Project description:Biogenesis of photosynthetic membranes depends on galactolipid synthesis, which relies on several cell compartments, notably the ER (endoplasmic reticulum) and the chloroplast envelope. Galactolipid synthesis involves lipid trafficking between both membrane compartments. In Arabidopsis, ALA10, a phospholipid flippase of the P4 type-ATPase family, has a positive effect on leaf development, interestingly counteracting the limitation of monogalactosyldiacylglycerol (MGDG) production. ALA10 locates in distinct domains of the ER depending on the ALIS (ALA interacting subunit) subunit it interacts with: close to the plasma membrane with ALIS1, or next to chloroplasts with ALIS5. It interacts with FAD2 (Fatty acid desaturase 2) and prevents accumulation of linolenic (18:3) containing phosphatidylcholine (PC) stimulating an increase of MGDG synthesis. Here we report that ALA10 interacts with PUB11 (plant U-box type 11), an E3 protein ubiquitin ligase, is ubiquitinated and degraded by the 26S proteasome in a PUB11 independent process.

2020-08-10 | PXD019412 | Pride

Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress

Project description:Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation (ChIP) analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention.

2020-04-24 | GSE131146 | GEO

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