Following the flux of long-chain bases through the sphingolipid pathway in vivo using mass spectrometry

Abstract

This article is available online at http://www.jlr.org sphingolipids, and plants can make of three classes of sphingolipids (3-6).Sphingolipids and their biosynthetic precursors, longchain base (LCB) and ceramide, have structural as well as signal functions; their synthesis and turnover thus must be precisely controlled (7). The rate-limiting step of the pathway is under control of serine palmitoyltransferase (SPT), an endoplasmic reticulum (ER)-localized protein complex that catalyzes the condensation of palmitoyl-CoA with serine to yield keto-dihydrosphingosine, a LCB (8). SPT activity is negatively regulated by the Orm proteins, conserved integral ER membrane proteins that form a protein complex together with the phosphoinositide phosphatase, Sac1 (5, 9, 10). Orm proteins are subject to phosphorylation and this relieves their inhibition of SPT activity (9). The kinases that phosphorylate the Orm proteins integrate multiple signals to maintain sphingolipid homeostasis, including heat, ER stress, and nutritional availability via the target of rapamycin (TOR) signaling network (11)(12)(13)(14)(15).After reduction of the 3-keto-dihydrosphingosine to dihydrosphingosine (DHS) and its further hydroxylation to phytosphingosine (PHS), DHS or PHS are converted to ceramide by ceramide synthase (CerS), which catalyzes the formation of an amide bond between the LCB and a C26 very long-chain fatty acid (16,17). CerS activity is regulated by direct phosphorylation of the catalytic subunits, Lac1 and Lag1, through TORC1, TORC2, and casein kinase 2 (see Fig. 1 for an overview of the pathway and the nomenclature of the intermediates) (15,(18)(19)(20).Ceramide is then converted in the Golgi to inositol phosphorylceramide ( Sphingolipids are greatly enriched in the plasma membrane of eukaryotic cells and they have been implicated in the formation of lateral membrane domains important for the spatial segregations of proteins during their transport to the plasma membrane (1, 2). Whereas animal cells produce sphingolipids in which the ceramide anchor contains a hydrophilic head group composed of choline or carbohydrate moieties, fungi synthesize inositol-containing

An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity

“…1 A). It has previously been shown that cells lacking either of these synthases have reduced levels of ceramides (Schorling et al, 2001;Rego et al, 2012;Martínez-Montañés and Schneiter, 2016). In contrast, cells lacking Orm1p, Orm2p, and Lcb4p, which phosphorylates sphingosine and other LCBs, remain hypersensitive to tunicamycin ( Fig.…”

Probing de novo sphingolipid metabolism in mammalian cells utilizing mass spectrometry

“…Moreover, assays that utilize thin layer chromatography for the detection of the labeled lipids are unable to discriminate the variations in the Nacyl chain length, which can play a role in sphingolipid biology [22][23][24]. One approach to dissecting cellular pools of lipids is to utilize a label such as an unnatural 17 carbon sphingoid base for flux analysis monitored by LC/MS/MS [25,26]. Assays of this nature have been utilized to assess CerS and SK activities measuring the conversion of d17sphingosine (d17Sph) into d17ceramide (d17Cer) or d17sphingosine-1-phosphate (d17S-1-P), respectively.…”

Tools for the analysis of metabolic flux through the sphingolipid pathway

“…Whereas wild-type cells respond in a dose-dependent manner to the addition of C17-PHS, Orm mutant cells display a reduction in ceramide and IPC synthesis if higher concentrations of C17-PHS are supplied [57].…”

“…In vitro assays to monitor sphingosine kinase or ceramide synthase activities by using non-naturally occurring C17-sphingosine or C17-DHS as substrates followed by product detection by mass spectrometry have been described previously [56]. More recently, administration of C17-PHS to living yeast cells was employed to monitor ceramide synthase and IPC production in vivo and yielded insights into the complex regulation of the pathway [57].…”