Yeast members of the ORMDL family of endoplasmic reticulum (ER) membrane proteins play a central role in lipid homeostasis and protein quality control. In the absence of yeast Orm1 and Orm2, accumulation of long chain base, a sphingolipid precursor, suggests dysregulation of sphingolipid synthesis. Physical interaction between Orm1 and Orm2 and serine palmitoyltransferase, responsible for the first committed step in sphingolipid synthesis, further supports a role for the Orm proteins in regulating sphingolipid synthesis. Phospholipid homeostasis is also affected in orm1 Δ orm2 Δ cells: the cells are inositol auxotrophs with impaired transcriptional regulation of genes encoding phospholipid biosynthesis enzymes. Strikingly, impaired growth of orm1 Δ orm2 Δ cells is associated with constitutive unfolded protein response, sensitivity to stress, and slow ER-to-Golgi transport. Inhibition of sphingolipid synthesis suppresses orm1 Δ orm2 Δ phenotypes, including ER stress, suggesting that disrupted sphingolipid homeostasis accounts for pleiotropic phenotypes. Thus, the yeast Orm proteins control membrane biogenesis by coordinating lipid homeostasis with protein quality control.

Sphingolipids (SLs) are structurally diverse lipids that are defined by the presence of a long-chain base (LCB) backbone. Typically, LCBs contain a single Δ4E double bond (DB) (mostly d18:1), whereas the dienic LCB sphingadienine (d18:2) contains a second DB at the Δ14Z position. The enzyme introducing the Δ14Z DB is unknown. We analyzed the LCB plasma profile in a gender-, age-, and BMI-matched subgroup of the CoLaus cohort (n = 658). Sphingadienine levels showed a significant association with gender, being on average ∼30% higher in females. A genome-wide association study (GWAS) revealed variants in the fatty acid desaturase 3 (FADS3) gene to be significantly associated with the plasma d18:2/d18:1 ratio (p = −log 7.9). Metabolic labeling assays, FADS3 overexpression and knockdown approaches, and plasma LCB profiling in FADS3-deficient mice confirmed that FADS3 is a bona fide LCB desaturase and required for the introduction of the Δ14Z double bond. Moreover, we showed that FADS3 is required for the conversion of the atypical cytotoxic 1-deoxysphinganine (1-deoxySA, m18:0) to 1-deoxysphingosine (1-deoxySO, m18:1). HEK293 cells overexpressing FADS3 were more resistant to m18:0 toxicity than WT cells. In summary, using a combination of metabolic profiling and GWAS, we identified FADS3 to be essential for forming Δ14Z DB containing LCBs, such as d18:2 and m18:1. Our results unravel FADS3 as a Δ14Z LCB desaturase, thereby disclosing the last missing enzyme of the SL de novo synthesis pathway.

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C₁₄ to C₁₈, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins. Significance Sphingolipids (SLs) are complex lipids that constitute hundreds of subspecies. All SLs share a long-chain base (LCB) as a defining structural component. LCBs are formed by serine-palmitoyltransferase (SPT) in the first and rate-limiting step of SL de novo synthesis. SPT consists of three subunits that show tissue-specific expression. In presence of the SPTLC3 subunit, the enzyme forms a spectrum of straight and branched LCBs with distinct biochemical and biophysical properties. This alters the composition of cellular membranes and might influence the dynamics of membrane-related transport and signaling events. SPTLC3 is particularly abundant in skin, and changes in SPT activity are related to dermal pathologies. Genetic variants of SPTLC3 are associated with metabolic conditions such as dyslipidemia and atherosclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons. Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids (SLs), cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased SL synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause peripheral hereditary sensory and autonomic neuropathy type 1 (HSAN1) due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS–expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.

Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously—Brr6, which is closely related to Brl1, and Apq12—function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE.