Fig 2: Model of SPT localization.Dual localization of SPTLC2 to the ER and mitochondria directs the SPT complex both to the ER and ER–mitochondria contact sites, respectively. SPTLC2 on the ER interacts in cis with ER-localized SPTLC1, whereas SPTLC2 on the mitochondrial outer membrane interacts in trans with SPTLC1 at membrane contact sites, providing mitochondria with ceramide precursors. SPTLC2 sensitizes cells to palmitate-induced fragmentation, and mitochondrial ceramide accumulation is linked to development of obesity and insulin resistance. Pathogenic SPT variants associated with neuropathy promote deoxy-sphingolipid synthesis.

Fig 3: Expression of WT-, ER-, and outer membrane (OM)-SPTLC2FLAG in A431 cells.(A) Purity of mitochondria used for determining newly synthesized mitochondrial sphingolipids isolated from A431 control, SPTLC2-KO, and SPTLC2-KO cells constitutively expressing WT-SPTLC2FLAG. Heavy membranes were separated on a Percoll-gradient from where MAMs (fraction 1) and mitochondria (fraction 3) were collected. Fractions were analyzed by SDS–PAGE and immunoblotting. (B) Confocal microscopy images of WT-, ER-, and OM-SPTLC2FLAG expression in A431 cells. Expression and localization of integrated constitutively expressed WT-, ER-, and OM-SPTLC2FLAG in SPTLC2-KO A431 cells (in BSA-treated control condition) was visualized using an anti-FLAG antibody. TOMM40 serves as a mitochondrial marker and Calnexin (CANX) as an ER marker. Scale bar 10 or 1 µm (zoomed image).Source data are available for this figure.

Fig 4: Mitochondrial SPTLC2 interacts with SPTLC1 in the ER.(A) Schematic of WT-, ER-, and OM-SPTLC2FLAG constructs. (B) Immunoblot analysis of SPTLC1 co-purification with WT-, ER-, and OM-SPTLC2FLAG. Heavy membrane fractions from Flp-In T-REx 293 SPTLC2-KO and SPTLC2-KO cells expressing WT-, ER-, and OM-SPTLC2FLAG were solubilized by digitonin, subjected to FLAG-immunoprecipitation, and input (5%) and eluate (25%) fractions were analyzed by SDS–PAGE and immunoblotting. (C) Super-resolution microscopy images of SPTLC2FLAG localization. SPTLC2FLAG was stably integrated into SPTLC2-KO U2OS cells and visualized by FLAG signal. ER-Emerald serves as ER marker and TOMM40 serves as mitochondrial marker. Orange circles highlight SPTLC2FLAG puncta at ER–mitochondria contact sites. Scale bar 2 or 0.5 µm (zoom). (D) Characterization of the SPT complex in mouse liver fractions. Light and heavy membrane fractions and purified mitochondria from mouse liver were analyzed by blue native PAGE. Samples were analyzed in duplicate on the same gel and immunoblotted with anti-SPTLC1 (left) and anti-SPTLC2 antibodies (right).Source data are available for this figure.

Fig 5: Analysis of SPTLC2FLAG localization and membrane association.(A) Subcellular localization of SPTLC2FLAG. Flp-In T-REx 293 SPTLC2-KO cells rescued with integrated tetracycline inducible SPTLC2FLAG were fractionated. Fractions were analyzed by SDS–PAGE and immunoblotting. (B) Submitochondrial localization of SPTLC2FLAG. Protease protection assay on crude mitochondria isolated from Flp-In T-REx 293 SPTLC2-KO cells rescued with integrated tetracycline inducible SPTLC2FLAG subjected to hypotonic swelling or solubilization with Triton X-100 (TX-100). Where indicated, samples were treated with proteinase K (PK). (C) Analysis of membrane association of SPTLC2FLAG by sodium carbonate extraction of heavy membrane fractions isolated from Flp-In T-REx 293 SPTLC2-KO cells rescued with integrated tetracycline inducible SPTLC2FLAG. Sup, supernatant; TM, transmembrane. (D) SPTLC2 KO and re-expression of integrated constitutively expressed SPTLC2FLAG in A431 cells. Whole cell lysates were analyzed on SDS–PAGE and immunoblotting.Source data are available for this figure.