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.

Figure 1.. SPTLC2 localizes to the ER and the mitochondrial outer membrane.

(A) Schematic of the SPT complex formed by SPTLC1 and SPTLC2 and its role in de novo sphingolipid synthesis. (B) Confocal microscopy images of SPTLC1 localization to the ER. SPTLC1-FLAG was transiently expressed in COS-7 cells and visualized using an anti-FLAG antibody. ER-mCherry serves as an ER marker and endogenous MRPL12 serves as a mitochondrial marker. Scale bar 10 or 1 μm (zoom). (C) Confocal microscopy images and line scan of fluorescence intensities demonstrating SPTLC2 localization to mitochondria and ER. SPTLC2-GFP was transiently expressed in COS-7 cells and visualized by GFP signal enhanced with anti-GFP-488 antibody. ER-mCherry serves as ER marker and MRPL12 serves as mitochondrial marker. Scale bar 10 or 1 μm (zoom). Line scan (from left to right) of fluorescence intensities normalized to total intensity of the channel. (D) Subcellular localization of SPTLC2 and SPTLC1 in vivo. Mouse liver was fractionated and the fractions were analyzed by SDS–PAGE and immunoblotting. (E) Submitochondrial localization of SPTLC2. Protease protection assay on crude mitochondria isolated from Flp-In T-REx 293 cells subjected to hypotonic swelling or solubilization with Triton X-100 (TX-100). Where indicated, samples were treated with proteinase K (PK). (F) Confocal microscopy images and line scan of fluorescence intensities demonstrating that the N-terminus of SPTLC2 targets mitochondria and ER. SPTLC2_1-102-GFP was transiently expressed in COS-7 cells and visualized by GFP signal enhanced with anti-GFP-488 antibody. ER-mCherry serves as an ER marker and MRPL12 serves as a mitochondrial marker. Scale bar 10 or 1 μm (zoom). Line scan (from left to right) of fluorescence intensities normalized to total intensity of the channel. Source data are available for this figure.

Figure S1.. N terminus of SPTLC2 is required for localization to the mitochondrial outer membrane.

(A) Confocal microscopy images demonstrating endogenous SPTLC1 localization to the ER. SPTLC1 in COS-7 cells was visualized with anti-SPTLC1 antibody, ER-mCherry (mCh-Sec61 beta) serves as an ER marker, and MRPL12 serves as a mitochondrial marker. Scale bar 10 or 1 μm (zoomed image). (B) Confocal microscopy images demonstrating endogenous DEGS1 localization to the mitochondria. DEGS1 in A431 cells was visualized with anti-DEGS1 antibody, CANX serves as an ER marker, and MRPL12 serves as a mitochondrial marker. Scale bar 10 μm. (C) Alignment of human SPTLC1 and SPTLC2 protein sequences. Alignment was generated using T-Coffee (Notredame et al, 2000) and BoxShade (https://github.com/mdbaron42/pyBoxshade). Predicted transmembrane sequences (according to CCTOP, http://cctop.enzim.ttk.mta.hu [Dobson et al, 2015]) are highlighted in magenta. Identical amino acids are highlighted in black and similar amino acids in gray. The conserved aminotransferase domain (pfam00155) is depicted in blue. (D) Confocal microscopy images and line scan of fluorescence intensities demonstrating SPTLC2_103-562-GFP localization to the cytosol. SPTLC2_103-562-GFP was transiently expressed in COS-7 cells and visualized by GFP signal enhanced with anti-GFP-488 antibody. ER-mCherry serves as an ER marker and MRPL12 serves as a mitochondrial marker. Scale bar 10 or 1 μm (zoomed image). Line scan (from left to right) of fluorescence intensities normalized to total intensity of the channel. (E) Analysis of membrane association of SPTLC1 and SPTLC2 by sodium carbonate extraction of heavy membrane fractions isolated from Flp-In T-Rex 293 cells. Sup, supernatant; TM, transmembrane. Source data are available for this figure.

Figure 2.. Analysis of SPTLC1-KO and SPTLC2-KO cells.

(A) SPTLC1 and SPTLC2 protein levels in SPTLC1-KO and SPTLC2-KO cells. Whole cell lysates from Flp-In T-REx 293 cells were analyzed by SDS–PAGE and immunoblotting. *, carryover signal from SPTLC2 decoration. Actin was used as a loading control. (A, B) Quantification of SPTLC1 (left panel) and SPTLC2 (right panel) protein levels in SPTLC1-KO (1-KO) and SPTLC2-KO (2-KO) from (A). SPTLC1 and SPTLC2 were normalized to Actin signal. Mean ± SD n = 3 independent replicates, unpaired two-sided Welch’s t test, *P < 0.05, **P < 0.01. (C) qRT-PCR analysis of SPTLC1 (left panel) and SPTLC2 (right panel) mRNA levels relative to control. Mean ± SD n = 3 independent replicates. (D) Characterization of the SPT complex. Heavy membrane fractions from Flp-In T-REx 293 cells, SPTLC-KOs and rescues 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). ATP5A antibody was used as a loading control. (E) Quantification of newly synthesized sphingolipids in cells. A431 control, SPTLC2-KO, and SPTLC2-KO cells constitutively expressing SPTLC2^FLAG were grown in the presence of (2,3,3-D3, ¹⁵N)-L-serine for 24 h. Lipids were extracted from cell pellets and stable isotope-labeled sphingolipids were analyzed by liquid chromatography–mass spectrometry. SPT-inhibitor myriocin was added for the duration of the labeling when indicated. Mean ± SD n = 3 independent replicates, unpaired two-sided t tests corrected for multiple comparisons, **P < 0.01, ***P < 0.001. (F) Immunoblot analysis of proteins co-purified with SPTLC2^FLAG. Heavy membrane fractions from Flp-In T-REx 293 SPTLC2-KO and SPTLC2-KO cells expressing SPTLC2^FLAG were solubilized by digitonin, subjected to FLAG-immunoprecipitation, and input (5%) and eluate (IP:FLAG, 40%) fractions were analyzed by SDS–PAGE and immunoblotting. SYNJ2BP was used as a negative control. Source data are available for this figure.

Figure S2.. Analysis of SPTLC2^FLAG localization and membrane association.

(A) Subcellular localization of SPTLC2^FLAG. Flp-In T-REx 293 SPTLC2-KO cells rescued with integrated tetracycline inducible SPTLC2^FLAG were fractionated. Fractions were analyzed by SDS–PAGE and immunoblotting. (B) Submitochondrial localization of SPTLC2^FLAG. Protease protection assay on crude mitochondria isolated from Flp-In T-REx 293 SPTLC2-KO cells rescued with integrated tetracycline inducible SPTLC2^FLAG 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 SPTLC2^FLAG by sodium carbonate extraction of heavy membrane fractions isolated from Flp-In T-REx 293 SPTLC2-KO cells rescued with integrated tetracycline inducible SPTLC2^FLAG. Sup, supernatant; TM, transmembrane. (D) SPTLC2 KO and re-expression of integrated constitutively expressed SPTLC2^FLAG in A431 cells. Whole cell lysates were analyzed on SDS–PAGE and immunoblotting. Source data are available for this figure.

Figure S3.. Mitochondrial SPTLC2 forms a complex with SPTLC1 in the ER.

(A) Confocal microscopy images of WT-, ER-, and outer membrane (OM)-SPTLC2^FLAG localization to mitochondria and ER. WT-, ER-, and OM-SPTLC2^FLAG were transiently expressed in COS-7 cells and visualized using an anti-FLAG antibody. ER-mCherry serves as an ER marker and endogenous CYCS serves as a mitochondrial marker. Scale bar 10 μm. (B) SPTLC2 KO and re-expression of integrated constitutively expressed SPTLC2^FLAG in U2OS cells. Whole cell lysates were analyzed on SDS–PAGE and immunoblotting. (C) Super-resolution microscopy images of SPTLC2^FLAG localization in A431 cells. SPTLC2^FLAG was stably integrated into SPTLC2-KO A431 cells and visualized by FLAG signal. ER-Emerald serves as ER marker and TOMM40 serves as mitochondrial marker. Scale bar 1 or 0.5 μm (zoom). (D) Expression levels of constitutively expressed WT-, ER-, and OM-SPTLC2^FLAG in A431 SPTLC2-KO cells. Whole cell lysates were analyzed on SDS–PAGE and immunoblotting. (E) SPT complex in SPTLC2-KO expressing WT-, ER-, and OM-SPTLC2^FLAG. Heavy membrane fractions from A431 cells were analyzed by blue native PAGE. Samples were analyzed in duplicate on the same gel and immunoblotted with anti-SPTLC2 (left) and anti-SPTLC1 (right) antibodies. ATP5A antibody was used as a loading control. (F) Quantification of newly synthesized sphingolipids in cells. A431 control, SPTLC2-KO, and SPTLC2-KO cells constitutively expressing WT-, ER-, and OM-SPTLC2^FLAG were grown in the presence of (2,3,3-D3, ¹⁵N)-L-serine for 24 h. Lipids were extracted from cell pellets and labeled sphingosine, ceramides and glycosphingolipids were analyzed by liquid chromatography–mass spectrometry. Data for CTRL, KO, and KO expressing SPTLC2^FLAG are the same as in Fig 2E. Mean ± SD n = 3 independent replicates, unpaired two-sided t tests corrected for multiple comparisons, **P < 0.01, ***P < 0.001. Source data are available for this figure.

Figure 3.. Mitochondrial SPTLC2 interacts with SPTLC1 in the ER.

(A) Schematic of WT-, ER-, and OM-SPTLC2^FLAG constructs. (B) Immunoblot analysis of SPTLC1 co-purification with WT-, ER-, and OM-SPTLC2^FLAG. Heavy membrane fractions from Flp-In T-REx 293 SPTLC2-KO and SPTLC2-KO cells expressing WT-, ER-, and OM-SPTLC2^FLAG 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 SPTLC2^FLAG localization. SPTLC2^FLAG 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 SPTLC2^FLAG 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.

Figure S4.. Expression of WT-, ER-, and outer membrane (OM)-SPTLC2^FLAG 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-SPTLC2^FLAG. 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-SPTLC2^FLAG expression in A431 cells. Expression and localization of integrated constitutively expressed WT-, ER-, and OM-SPTLC2^FLAG 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.

Figure 4.. Lack of mitochondrial SPTLC2 protein attenuates palmitate induced mitochondrial fragmentation.

(A) Quantification of newly synthesized sphingolipids in mitochondria. A431 control, SPTLC2-KO, and SPTLC2-KO cells constitutively expressing WT-SPTLC2^FLAG were grown in the presence of (2,3,3-D3, ¹⁵N)-L-serine for 24 h. Mitochondria were isolated by gradient-purification and stable isotope-labeled sphingolipids were extracted and analyzed by liquid chromatography–mass spectrometry. Mean ± SD n = 3 independent replicates, unpaired two-sided Welch’s t test, *P < 0.05. (B) Representative confocal images of mitochondrial morphology in A431 cells by TOMM40 immunofluorescence after 8 h control (BSA) or palmitate (BSA:PALMITATE) treatment. A431 control, SPTLC2-KO, and SPTLC2-KO cells constitutively expressing WT-, ER-, or OM-SPTLC2^FLAG. Scale bar 10 μm. (C) Quantification of cells with a fragmented mitochondrial network. 125–269 cells were counted from 19 to 34 images/genotype/experiment. Mean ± SD n = 5 independent replicates, unpaired two-sided t test, **P < 0.01, ***P < 0.001, ns, not significant. (D) Representative confocal images of mitochondrial morphology in A431 control cells by TOMM40 immunofluorescence after 8 h palmitate treatment in the absence or presence of myriocin. Scale bar 10 μm. (E) Quantification of cells with a fragmented mitochondrial network. 231–367 cells were counted from 26 to 30 images/condition/experiment. Mean ± SD n = 3 independent replicates, unpaired two-sided t test, *P < 0.05. Source data are available for this figure.

Figure 5.. 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.