analysis-neutral-lipid-synthesis-saccharomyces-cerevisiae-metabolic

Analysis of Neutral Lipid Synthesis in <em>Saccharomyces cerevisiae</em> by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

University of Texas Southwestern Medical Center

Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, we conducted a global fluorescence-based screen to reveal endomembrane effector genes. Screening implicated Phox (PX) domain-containing protein Mdm1 in endomembrane dynamics. Surprisingly, we demonstrate that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)-vacuole/lysosome membrane contact sites (MCSs). We show that Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER-vacuole hypertethering, underscoring its role as an interorganelle tether. We also show that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER-vacuole MCSs independently of established tether Nvj1. Finally, we find that Mdm1 truncations analogous to neurological disease-associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. Our work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism.

Mdm1/Snx13 is a novel ER-endolysosomal interorganelle tethering protein.

Cellular organelles must execute sophisticated biological processes to persist, and often communicate with one another to exchange metabolites and information. Recent studies suggest inter-organelle membrane contact sites (MCSs) are hubs for this cellular cross-talk. MCSs also govern membrane remodeling, thus controlling aspects of organelle shape, identity, and function. Here, we summarize three emerging phenomena that MCSs appear to govern: 1) organelle identity via the non-vesicular exchange of lipids, 2) mitochondrial shape and division, and 3) endosomal migration in response to sterol trafficking. We also discuss the role for ER-endolysosomal contact sites in cholesterol metabolism, and the potential biomedical importance this holds. Indeed, the emerging field inter-organellar cross-talk promises substantial advances in the fields of lipid metabolism and cell signaling.

Organelle remodeling at membrane contact sites.

Inter-organelle membrane contact sites (MCSs) serve as unique microenvironments for the sensing and exchange of cellular metabolites and lipids. Though poorly defined, ER-endolysosomal contact sites are quickly becoming recognized as centers for inter-organelle lipid exchange and metabolic decision-making. Here, we review the discovery and current state of knowledge of ER-endolysosomal MCSs with particular focus on the molecular players that establish and/or utilize these contact sites in metabolism. We also discuss associations of ER-endolysosomal MCS-associated proteins in human disease, as well as the therapeutic promise these contact sites hold in modulating cellular physiology.

Discovery and Roles of ER-Endolysosomal Contact Sites in Disease.

Eukaryotic cells store lipids in cytosolic organelles known as lipid droplets (LDs). Lipid droplet bud from the endoplasmic reticulum (ER), and may be harvested by the vacuole for energy during prolonged periods of starvation. How cells spatially coordinate LD production is poorly understood. Here, we demonstrate that yeast ER-vacuole contact sites (NVJs) physically expand in response to metabolic stress, and serve as sites for LD production. NVJ tether Mdm1 demarcates sites of LD budding, and interacts with fatty acyl-CoA synthases at the NVJ periphery. Artificially expanding the NVJ through over-expressing Mdm1 is sufficient to drive NVJ-associated LD production, whereas ablating the NVJ induces defects in fatty acid-to-triglyceride production. Collectively, our data suggest a tight metabolic link between nutritional stress and LD biogenesis that is spatially coordinated at ER-vacuole contact sites.

Lipid droplet biogenesis is spatially coordinated at ER-vacuole contacts under nutritional stress.

In mammals, blood glucose levels likely play a role in appetite regulation yet the mechanisms underlying this phenomenon remain opaque. Mechanisms can often be explored from  genetic approaches. To determine if circulating sugars might be involved in  feeding behaviors, we scored hemolymph glucose and trehalose, and food ingestion in larvae subjected to various diets, genetic mutations, or RNAi. We found that larvae with glucose elevations, hyperglycemia, have an aversion to feeding; however, trehalose levels do not track with feeding behavior. We further discovered that insulins and SLC5A11 may participate in glucose-regulated feeding. To see if food aversion might be an appropriate screening method for hyperglycemia candidates, we developed a food aversion screen to score larvae with abnormal feeding for glucose. We found that many feeding defective larvae have glucose elevations. These findings highlight intriguing roles for glucose in fly biology as a potential cue and regulator of appetite.

Circulating glucose levels inversely correlate with  larval feeding through insulin signaling and SLC5A11.

Lipid droplets (LDs) are nutrient reservoirs used by cells to maintain homeostasis. Nascent droplets form on the endoplasmic reticulum (ER) and grow following an influx of exogenous fatty acids (FAs). The budding of LDs requires extensive ER-LD crosstalk, but how this is regulated remains poorly understood. Here, we show that sorting nexin protein Snx14, an ER-resident protein associated with the cerebellar ataxia SCAR20, localizes to ER-LD contacts following FA treatment, where it promotes LD maturation. Using proximity-based APEX technology and topological dissection, we show that Snx14 accumulates specifically at ER-LD contacts independently of Seipin, where it remains ER-anchored and binds LDs in trans.  cells exhibit perturbed LD morphology, whereas Snx14 overexpression promotes LD biogenesis and extends ER-LD contacts. Multi-time point imaging reveals that Snx14 is recruited to ER microdomains containing the fatty acyl-CoA ligase ACSL3, where nascent LDs bud. We propose that Snx14 is a novel marker for ER-LD contacts and regulates FA-stimulated LD growth.

Cerebellar ataxia disease-associated Snx14 promotes lipid droplet growth at ER-droplet contacts.

Lipid droplets (LDs) serve as cytoplasmic reservoirs for energy-rich fatty acids (FAs) stored in the form of triacylglycerides (TAGs). During nutrient stress, yeast LDs cluster adjacent to the vacuole/lysosome, but how this LD accumulation is coordinated remains poorly understood. The ER protein Mdm1 is a molecular tether that plays a role in clustering LDs during nutrient depletion, but its mechanism of function remains unknown. Here, we show that Mdm1 associates with LDs through its hydrophobic N-terminal region, which is sufficient to demarcate sites for LD budding. Mdm1 binds FAs via its Phox-associated domain and coenriches with fatty acyl-coenzyme A ligase Faa1 at LD bud sites. Consistent with this, loss of  perturbs free FA activation and Dga1-dependent synthesis of TAGs, elevating the cellular FA level, which perturbs ER morphology and sensitizes yeast to FA-induced lipotoxicity. We propose that Mdm1 coordinates FA activation adjacent to the vacuole to promote LD production in response to stress, thus maintaining ER homeostasis.

Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis.

Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3P, others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3P, non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family.

Classification of the human phox homology (PX) domains based on their phosphoinositide binding specificities.

Lipid droplets (LDs) store energy-rich fatty acids (FAs), but to harvest them, LDs donate FAs to peroxisomes for oxidation. In this issue, Chang et al. (2019.  https://doi.org/10.1083/jcb.201902061) identify M1 Spastin as an LD-peroxisome tether that interacts with ESCRT-III components to facilitate lipid exchange between these two organelles.

Spastin joins LDs and peroxisomes in the interorganelle contact ballet.

Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Here, using Drosophila fat body (FB) adipocytes, we characterize spatially distinct LD populations that are maintained by different lipid pools. We identify peripheral LDs (pLDs) that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. We find that sorting nexin CG1514 or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of SNZ perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a DESAT1-dependent pathway. We propose that Drosophila adipocytes maintain spatially distinct LD populations and identify Snz as a regulator of LD organization and inter-organelle crosstalk.

Drosophila Snazarus Regulates a Lipid Droplet Population at Plasma Membrane-Droplet Contacts in Adipocytes.

Isogenic cells manifest distinct cellular fates for a single stress; however, the nongenetic mechanisms driving such fates remain poorly understood. Here, we implement a robust multi-channel live-cell imaging approach to uncover noncanonical factors governing cell fate. We show that in response to acute glucose removal (AGR), budding yeast undergoes distinct fates, becoming either quiescent or senescent. Senescent cells fail to resume mitotic cycles following glucose replenishment but remain responsive to nutrient stimuli. Whereas quiescent cells manifest starvation-induced adaptation, senescent cells display perturbed endomembrane trafficking and defective nucleus-vacuole junction (NVJ) expansion. Surprisingly, senescence occurs even in the absence of lipid droplets. Importantly, we identify the nutrient-sensing kinase Rim15 as a key biomarker predicting cell fates before AGR stress. We propose that isogenic yeast challenged with acute nutrient shortage contains determinants influencing post-stress fate and demonstrate that specific nutrient signaling, stress response, trafficking, and inter-organelle biomarkers are early indicators for long-term fate outcomes.

Nutrient Signaling, Stress Response, and Inter-organelle Communication Are Non-canonical Determinants of Cell Fate.

Fatty acids (FAs) are central cellular metabolites that contribute to lipid synthesis, and can be stored or harvested for metabolic energy. Dysregulation in FA processing and storage causes toxic FA accumulation or altered membrane compositions and contributes to metabolic and neurological disorders. Saturated lipids are particularly detrimental to cells, but how lipid saturation levels are maintained remains poorly understood. Here, we identify the cerebellar ataxia spinocerebellar ataxia, autosomal recessive 20 (SCAR20)-associated protein Snx14, an endoplasmic reticulum (ER)-lipid droplet (LD) tethering protein, as a factor required to maintain the lipid saturation balance of cell membranes. We show that following saturated FA (SFA) treatment, the ER integrity of  cells is compromised, and both  cells and SCAR20 disease patient-derived cells are hypersensitive to SFA-mediated lipotoxic cell death. Using APEX2-based proximity labeling, we reveal the protein composition of Snx14-associated ER-LD contacts and define a functional interaction between Snx14 and Δ-9 FA desaturase SCD1. Lipidomic profiling reveals that  cells increase membrane lipid saturation following exposure to palmitate, phenocopying cells with perturbed SCD1 activity. In line with this,  cells manifest delayed FA processing and lipotoxicity, which can be rescued by SCD1 overexpression. Altogether, these mechanistic insights reveal a role for Snx14 in FA and ER homeostasis, defects in which may underlie the neuropathology of SCAR20.

Article	Year
Mdm1/Snx13 is a novel ER-endolysosomal interorganelle tethering protein. The Journal of cell biology\| PubMed ID: 26283797	2015
Organelle remodeling at membrane contact sites. Journal of structural biology\| PubMed ID: 27181417	2016
Discovery and Roles of ER-Endolysosomal Contact Sites in Disease. Advances in experimental medicine and biology\| PubMed ID: 28815527	2017
Lipid droplet biogenesis is spatially coordinated at ER-vacuole contacts under nutritional stress. EMBO reports\| PubMed ID: 29146766	2018
Circulating glucose levels inversely correlate with larval feeding through insulin signaling and SLC5A11. Communications biology\| PubMed ID: 30271990	2018
Cerebellar ataxia disease-associated Snx14 promotes lipid droplet growth at ER-droplet contacts. The Journal of cell biology\| PubMed ID: 30765438	2019
Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis. The Journal of cell biology\| PubMed ID: 30808705	2019
Classification of the human phox homology (PX) domains based on their phosphoinositide binding specificities. Nature communications\| PubMed ID: 30948714	2019
Spastin joins LDs and peroxisomes in the interorganelle contact ballet. The Journal of cell biology\| PubMed ID: 31278079	2019
Drosophila Snazarus Regulates a Lipid Droplet Population at Plasma Membrane-Droplet Contacts in Adipocytes. Developmental cell\| PubMed ID: 31422916	2019
Nutrient Signaling, Stress Response, and Inter-organelle Communication Are Non-canonical Determinants of Cell Fate. Cell reports\| PubMed ID: 33264609	2020
Snx14 proximity labeling reveals a role in saturated fatty acid metabolism and ER homeostasis defective in SCAR20 disease. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 33310904	2020

W. Mike Henne

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