Abstract
Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually.
Highlights
Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM)
In postnuclear supernatants of yeast cells expressing SMS2, both NBD-SM and NBD-ceramide phosphoethanolamine (CPE) were formed (Fig. 2A), indicating that this enzyme possesses dual activity. These results suggest that the SM synthase family comprises both monoand bifunctional enzymes, with each enzyme displaying a distinct substrate specificity
We show that the mammalian SMS family contains both single and dual activity enzymes, with SMS1 being a monofunctional SM synthase, SMSr a monofunctional CPE synthase, and SMS2 a bifunctional enzyme with both SM and CPE synthase activity
Summary
Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). A total of 100 OD of 4⌬.CerS5 yeast cells expressing human SMS1, SMS2, SMSr, or control insert was washed with water, treated with trichloroacetic acid to inactivate lipases, and boiled for 10 min [17] before lipid extraction by bead bashing in chloroform-methanol-water (10:10:3, v/v/v) [18]. Human SMS1, SMS2, and SMSr were expressed in budding yeast, and their enzymatic activities were investigated in vitro by incubating postnuclear supernatants with fluorescently labeled ceramide (NBD-Cer) as described in Experimental Procedures.
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