Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle

Abstract

One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers.sphingomyelin | Golgi apparatus | secretion | equinatoxin A mple evidence indicates that proteins are sorted in the the trans Golgi network (TGN) into distinct types of Golgiderived transport carriers (1), but little is known regarding the lipid content of different carriers. The most abundant sphingolipid, sphingomyelin (SM), is a principal component of the plasma membrane that is synthesized on the luminal membrane leaflets of TGN membranes and transported to the plasma membrane via an uncharacterized pathway. Inhibition of SM synthesis has been reported to slow Golgi-to-plasma membrane trafficking of vesicular stomatitis virus G protein, influenza hemagglutinin, and pancreatic adenocarcinoma up-regulated factor (2-6), suggesting that the SM biosynthetic pathway is broadly required for secretory competence, but the underlying mechanisms are unknown. Furthermore, it remains unclear whether SM trafficking per se, or the activities of SM metabolites such as ceramide and diacylglycerol (DAG), are harnessed for the production of secretory vesicles.Many investigations of intracellular sphingolipid sorting use synthetic short-chain ceramides that are labeled with a fluorescent moiety that can be metabolized, albeit at slow, nonphysiological rates, to short-chain fluorescent SM and glucosylceramide (7-9). In one of the first studies of SM sorting in a polarized epithelial cell line incubated with fluorescent short-chain ceramide, fluorescently labeled lipids accumulated to a higher level in the apical membrane domain compared with the basolateral domain, suggesting that the fluorescently labeled sphingolipids are enriched in apically targeted secretory vesicles (9). A study of secretory vesicle lipid content of yeast (Saccharomyces cerevisiae) cells, which produce mannosylated sphingolipids (but not SM) and ergosterol (but not cholesterol), found that two types of immunopurified secretory vesicles do not differ in terms of abundances of different lipid species (10, 11). Thus, the extent to which lipid sorting occurs in the TGN ...

Sphingolipids and Lipoproteins in Health and Metabolic Disorders

“…In the cis Golgi, sphingomyelin synthase 1 (SMS1) transfers the phosphorylcholine group from phosphatidylcholine to ceramide to form sphingomyelin (Fig 2B). Sphingomyelin is transported to the plasma membrane in distinct vesicles, where it forms lipid rafts with cholesterol 9 .…”

Sphingolipid abnormalities in cancer multidrug resistance: Chicken or egg?

“…Two isoforms of SMS ( SGMS1 and SGMS2 ) are localized to Golgi. Similar to proteins, SM is trafficked to the plasma membrane (PM) in a subset of specialized Golgi-derived secretory vesicles [13]. Moreover, trafficking processes exhibit dependence on SM [14, 15].…”

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

“…Once at the TGN, SLs and cholesterol are incorporated in membrane carriers and transported to post-Golgi destinations, thus contributing to the establishment of the post-Golgi membrane territory (De Matteis & Luini, 2008;Klemm et al, 2009;Deng et al, 2016). Nevertheless, carriers formation at the TGN occurs continuously so that TGN membranes are subjected to constant dissipation and rebuilding according to secretory flux volume to the TGN (De Matteis & Luini, 2008).…”