Alignment was generated using T-Coffee (Notredame et al, 2000) and BoxShade (https://github

Alignment was generated using T-Coffee (Notredame et al, 2000) and BoxShade (https://github.com/mdbaron42/pyBoxshade). we show that SPTLC2, a subunit of the serine palmitoyltransferase (SPT) complex, catalyzing the Histone Acetyltransferase Inhibitor II 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 ERCmitochondria contact sites. Introduction The lipid composition of cellular membranes is determined by the specific localization of proteins mediating lipid synthesis and transport. Most lipids are synthesized in the ER and need to be transported to other organelles via vesicles or by lipid transfer proteins at membrane contact sites. Contact sites are essential for pathways and functions requiring organellar communication. For example, proteins from the mitochondrial outer membrane (OM) and mitochondria-associated ER membrane (MAM) interact to allow exchange of molecules, such as phospholipids, at ERCmitochondria contact sites (Scorrano et al, 2019; Prinz et al, 2020). Serine palmitoyltransferase (SPT) catalyzes the first and rate-limiting step of de novo sphingolipid synthesis by producing 3-keto-sphinganine from L-serine and palmitoyl-CoA (Fig 1A) (Merrill & Wang, 1986). Via a multi-enzymatic cascade, this sphingoid base is converted to ceramide through the addition of an N-acyl chain, and further converted to a multitude of sphingolipid species, including sphingosine, Rabbit Polyclonal to Akt1 (phospho-Thr450) glycosphingolipids, and gangliosides (Pruett et al, 2008). The SPT core complex is formed by two subunits: SPTLC1 and the cofactor pyridoxal-phosphate made up of SPTLC2, or its homolog SPTLC3, and presence of both subunits is essential for enzyme activity (Lone et al, 2020). SPT formed by ubiquitously expressed SPTLC1 and SPTLC2 preferably synthesizes the palmitoyl-containing sphingoid base (Lone et al, 2020), which is the most abundant sphingoid base in mammalian cells (Pruett et al, 2008), whereas SPTLC3 expression is restricted to specific tissues and leads to the production of short-, long-, and branched-chain sphingolipid species (Hornemann et al, 2006, 2009; Lone et al, 2020). Open in a separate window Physique 1. SPTLC2 localizes to the ER and the mitochondrial outer membrane.(A) Schematic of the SPT complex formed by SPTLC1 and SPTLC2 and its Histone Acetyltransferase Inhibitor II 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 m (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 Histone Acetyltransferase Inhibitor II 10 or 1 m (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 SDSCPAGE 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. SPTLC21-102-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 m (zoom). Line scan (from left to right) of fluorescence intensities normalized to total intensity of the channel. Source data are available for this figure. Mutations in and cause hereditary sensory and autonomic neuropathy type 1, a rare neurological disease (Dawkins et al, 2001; Rotthier et al,.