Serine palmitoyltransferase assembles at ER–mitochondria contact sites

Abstract

The accumulation of sphingolipid species in the cell contributes to the development of obesity and neurological disease. However, the subcellular localization of sphingolipid-synthesizing enzymes is unclear, limiting the understanding of where and how these lipids accumulate inside the cell and why they are toxic. Here, we show that SPTLC2, a subunit of the serine palmitoyltransferase (SPT) complex, catalyzing the first step in de novo sphingolipid synthesis, localizes dually to the ER and the outer mitochondrial membrane. We demonstrate that mitochondrial SPTLC2 interacts and forms a complex in trans with the ER-localized SPT subunit SPTLC1. Loss of SPTLC2 prevents the synthesis of mitochondrial sphingolipids and protects from palmitate-induced mitochondrial toxicity, a process dependent on mitochondrial ceramides. Our results reveal the in trans assembly of an enzymatic complex at an organellar membrane contact site, providing novel insight into the localization of sphingolipid synthesis and the composition and function of ER–mitochondria contact sites.

SPTLC1 variants associated with childhood onset amyotrophic lateral sclerosis produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

“…For the biochemical analysis of SPTLC1 variants, C-terminally FLAG-tagged WT-SPTLC1 and C133W, S331F, Y23F, L39del, F40S41del and ex2del variants, were integrated into 293 Flp-In T-REx SPTLC1-KO cells (Fig 1C). The stability of SPTLC2 depends on SPTLC1 (15, 21) and the diminished level of SPTLC2 in SPTLC1-KO cells (17 % of control) was rescued upon re-expression of WT, C133W, S331F and Y23F variants, while a partial rescue was observed by L39del (77 % of control), F40S41del (68 % of control) and ex2del (38 % of control) variants (Fig 1C-D).…”

“…The generation of HEK293-SPTLC1 knockout cell line (for lipidomics) was reported earlier (4). SPTLC1-KO and SPTLC2-KO Flp-In T-Rex 293 cell lines (protein analysis) were reported before (15). Patient fibroblasts were cultured in DMEM with 10% FBS as reported previously (4).…”

“…pcDNA5-pDEST-SPTLC1 FLAG plasmid was generated by flipping SPTLC1 from pDONR221 donor vector into pcDNA5-pDEST-3xFLAG destination vector (15) using Gateway cloning technology, yielding SPTLC1 with C-terminal triple FLAG-tag. SPTLC1 variants were generated using mutagenesis PCR (QuikChange Lightning, Agilent) in pDONR221-SPTLC1 and flipped into pcDNA5-pDEST-3xFLAG destination vector.…”

“…(10)(11)(12)(13). Although the majority of reported SPTLC1-HSAN1variants are associated with sensory symptoms, a significant motor involvement was reported for the two variants, SPTLC1-S331F/Y (14).The SPT enzyme complex resides in the endoplasmic reticulum (ER) membrane and at the ER-mitochondrial contact sites (15). SPT is typically composed of two SPTLC1-SPTLC2 dimers which interact with the accessory subunits ssSPTa/b and the regulatory ORMDL proteins ORMDL1, -2 and -3, which are paralogous and functionally redundant proteins (16,17).…”

IMC10 and LMF1 mediate membrane contact between the mitochondrion and the inner membrane complex in Toxoplasma gondii

“…Membrane contact sites (MCS) are defined as regions where membranes from two compartments are tethered in close apposition (∼30 nm) and in which specific proteins and/or lipids are enriched (Eisenberg-Bord et al, 2016; Prinz, 2014). These contact sites are important for several physiological processes, including ion (Raturi et al, 2016) and lipid exchange (Aaltonen et al, 2022). In Toxoplasma, contact between organelles has been described between the mitochondrion and the apicoplast (Nishi et al, 2008), the endoplasmic reticulum (Mallo et al, 2021), the IMC (Jacobs et al, 2020; Ovciarikova et al, 2017) and between the ER and the apicoplast (Tomova et al, 2009).…”