The structure of the Orm2-containing serine palmitoyltransferase complex reveals distinct inhibitory potentials of yeast Orm proteins.

Sphingolipid levels are crucial determinants of neurodegenerative disorders and therefore require tight regulation. The Orm protein family and ceramides inhibit the rate-limiting step of sphingolipid biosynthesis-the condensation of L-serine and palmitoyl-coenzyme A (CoA). The yeast isoforms Orm1 and Orm2 form a complex with the serine palmitoyltransferase (SPT). While Orm1 and Orm2 have highly similar sequences, they are differentially regulated, though the mechanistic details remain elusive. Here, we determine the cryoelectron microscopy structure of the SPT complex containing Orm2. Complementary in vitro activity assays and genetic experiments with targeted lipidomics demonstrate a lower activity of the SPT-Orm2 complex than the SPT-Orm1 complex. Our results suggest a higher inhibitory potential of Orm2, despite the similar structures of the Orm1- and Orm2-containing complexes. The high conservation of SPT from yeast to man implies different regulatory capacities for the three human ORMDL isoforms, which might be key for understanding their role in sphingolipid-mediated neurodegenerative disorders.

Ferroptosis-protective membrane domains in quiescence.

Quiescence is a common cellular state, required for stem cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging and defective quiescence maintenance and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFAs). FLPs are essential for the extramitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work describes ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor in the long-term survival of quiescent cells.

Identification of distinct active pools of yeast serine palmitoyltransferase in sub-compartments of the ER.

Sphingolipids (SPs) are one of the three major lipid classes in eukaryotic cells and serve as structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is catalyzed by the serine palmitoyltransferase (SPT). In budding yeast (Saccharomyces cerevisiae), SPT is negatively regulated by the two proteins, Orm1 and Orm2. Regulating SPT activity enables cells to adapt SP metabolism to changing environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling pathways originating from either the plasma membrane or the lysosome (or vacuole in yeast). Moreover, uptake of exogenous serine is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially regulated SPT pools based on their intracellular localization. However, measuring lipid metabolic enzyme activity in different cellular sub-compartments has been challenging. Combining a nanobody recruitment approach with SP flux analysis, we show that the nuclear endoplasmic reticulum (ER)-localized SPT and the peripheral ER localized SPT pools are differentially active. Thus, our data add another layer to the complex network of SPT regulation. Moreover, combining lipid metabolic enzyme re-localization with flux analysis serves as versatile tool to measure lipid metabolism with subcellular resolution.

Structure of the ceramide-bound SPOTS complex.

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (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.

A lysosomal biogenesis map reveals the cargo spectrum of yeast vacuolar protein targeting pathways.

The lysosome is the major catabolic organelle in the cell that has been established as a key metabolic signaling center. Mutations in many lysosomal proteins have catastrophic effects and cause neurodegeneration, cancer, and age-related diseases. The vacuole is the lysosomal analog of Saccharomyces cerevisiae that harbors many evolutionary conserved proteins. Proteins reach vacuoles via the Vps10-dependent endosomal vacuolar protein sorting pathway, via the alkaline phosphatase (ALP or AP-3) pathway, and via the cytosol-to-vacuole transport (CVT) pathway. A systematic understanding of the cargo spectrum of each pathway is completely lacking. Here, we use quantitative proteomics of purified vacuoles to generate the yeast lysosomal biogenesis map. This dataset harbors information on the cargo-receptor relationship of almost all vacuolar proteins. We map binding motifs of Vps10 and the AP-3 complex and identify a novel cargo of the CVT pathway under nutrient-rich conditions. Our data show how organelle purification and quantitative proteomics can uncover fundamental insights into organelle biogenesis.

Uptake of exogenous 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 gnp1Δ 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.