Sphingolipid metabolic flow controls phosphoinositide turnover at the trans ‐Golgi network

Abstract

Abstract:Sphingolipids are membrane lipids, which are globally required for eukaryotic life.Sphingolipid composition varies among endomembranes with pre-and post-Golgi compartments being poor and rich in sphingolipids, respectively. Thanks to this different sphingolipid content, pre-and post-Golgi membranes serve different cellular functions.Nevertheless, how subcellular sphingolipid levels are maintained in spite of trafficking and metabolic fluxes is only partially understood. Here we describe a homeostatic control circuit that controls sphingolipid levels at the trans Golgi network. Specifically, we show that sphingomyelin production at the trans Golgi network triggers a signalling reaction leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol, and sphingolipid transport to the trans Golgi network, PtdIns(4)P consumption leads to the interruption of this transport in response to excessive sphingomyelin production. Based on this evidence we envisage a model where this homeostatic circuit maintains the lipid composition of trans Golgi network and thus of post-Golgi compartments constant, against instant fluctuations in the sphingolipid biosynthetic flow. Introduction:

Probing de novo sphingolipid metabolism in mammalian cells utilizing mass spectrometry

“…Based on rates of label incorporation into 17 carbon-sphingolipid metabolites during a 120 min precursor pulse, we identified discrete phases of synthesis of sphingolipid metabolites that correspond to the enzymatic activities for CerS, DeS, HexCer, and SM synthesis in MCF-7 breast cancer cells. These kinetic results also agree with what is currently known about the subcellular localization of de novo synthesis of sphingolipids [36,[38][39][40]. Notably, all results were acquired from a single-step approach utilizing a 17 carbon-sphingoid label that demonstrates very similar biochemical properties to the endogenous substrates; as such, all obtained metabolic rates are comparable to those of natural sphingolipids.…”

“…Our data demonstrate that generation of d17SM and d17HexCer occur at relatively the same rate and with similar initial incorporation of our label (Fig 6). Thus, utilizing it has been reported that BFA treatment enhanced incorporation of short chain ceramides into short chain SM while the conversion into short chain HexCer was unchanged [39,49]. In our study, BFA decreased the time the d17Cer took to reach SMS enzymes from 35 min to 5 min suggesting that 30 min is approximately the time necessary for Cer to reach SMS in the Golgi from the ER (Fig 6E).…”

“…BFA is an antibiotic that has been shown to induce fusion of the Golgi and ER [38], producing an increase in sphingomyelin synthase (SMS) activity while having lesser effects on HexCer synthesis [39]. MCF-7 cells were pretreated with a 24μM dose of BFA for 1 hour, and d17-labeled sphingolipids were assessed with ESI/MS/MS.…”

Structure, functions and regulation of CERT, a lipid‐transfer protein for the delivery of ceramide at the ER–Golgi membrane contact sites

“…3). D'Angelo et al recently showed that the sustained sphingolipid inflow into the trans-Golgi regions reduces the level of PtdIns(4)P at the trans-Golgi regions, which causes the down-regulation of Golgi-targeting of CERT and consequently represses SM biosynthesis [64]. The reduction in PtdIns(4)P level is dependent on SMS, PKD, OSBP and SAC1 [64].…”

“…D'Angelo et al recently showed that the sustained sphingolipid inflow into the trans-Golgi regions reduces the level of PtdIns(4)P at the trans-Golgi regions, which causes the down-regulation of Golgi-targeting of CERT and consequently represses SM biosynthesis [64]. The reduction in PtdIns(4)P level is dependent on SMS, PKD, OSBP and SAC1 [64]. When a water-miscible short-chain ceramide was exogenously added to cultured cells, it was converted to short-chain SM in the Golgi complex by SMS with an equimolar generation of DAG in a CERT-independent manner.…”