Limar, S. | bioRxiv Science

bioRxiv2026

Lipid Droplet Targeting Drives Substrate Access during Very Long Chain Fatty Acid Activation by Fat1

Fatty acids, the most abundant building blocks for lipid synthesis, require conjugation with Coenzyme A (CoA) for their incorporation into membrane lipids. Of those, very long chain fatty acids (VLCFAs), the precursors of yeast ceramides, are particularly hydrophobic. Yet it remains unresolved where and how the sole VLCFA-CoA synthetase Fat1 activates them. Here we show that lipid droplets (LDs) are both the site of free VLCFAs storage, as well as their place of activation. Free VLCFAs preferentially partition into LDs in molecular simulations and in vitro reconstitutions, whereas Fat1 targets the LD surface in cells via a N-terminal amphipathic helix. Structural predictions identify an essential hydrophobic cavity in Fat1 that connects the active site to the membrane, suggesting that VLCFAs directly shuttle from the LD core into the enzymatic pocket. Our model explains how the organelle targeting of a fatty acid activator evolved to match the biophysical properties and consequently the subcellular localization of its highly hydrophobic substrate.

biochemistry↗

bioRxiv2023

Structure of the ceramide-bound SPOTS complex

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyl-transferase (SPT) catalyzes the rate limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.

biochemistry↗

bioRxiv2020

Uptake of endogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1{Delta} cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism. Author SummarySphingolipids (SPs) are membrane lipids globally required for eukaryotic life. In contrast to other lipid classes, SPs cannot be stored in the cell and therefore their levels have to be tightly regulated. Failure to maintain sphingolipid homeostasis can result in pathologies including neurodegeneration, childhood asthma and cancer. However, we are only starting to understand how SP biosynthesis is adjusted according to need. In this study, we use genetic and biochemical methods to show that the uptake of exogenous serine is necessary to maintain SP homeostasis in Saccharomyces cerevisiae. Serine is one of the precursors of long chain bases in cells, the first intermediate of SP metabolism. Our results suggest that the uptake of serine is directly coupled to SP biosynthesis at ER-plasma membrane contact sites. Overall, our study identifies serine uptake as a novel regulatory factor of SP homeostasis. While we use yeast as a discovery tool, these results also provide valuable insights into mammalian SP biology especially under pathological conditions.

genetics↗