Abstract
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.
Highlights
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting
Heterologous expression of these constructs in HeLa cells followed by immunoblot analysis in each case yielded a gel-shifted EndoH-sensitive protein that was absent in SMS-related protein (SMSr)-V5-expressing cells (Fig. 1B)
When lysates of yeast cells expressing SMS2 were incubated with the fluorescent ceramide analog, NBD-Cer, both NBD-SM and NBD-ceramide phosphoethanolamine (CPE) were formed, in line with previous studies indicating that this enzyme possesses dual SM and CPE synthase activity (Fig. 1D) [19]
Summary
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. While the biological relevance of SMS2-mediated CPE production remains to be established, acute disruption of SMSr catalytic activity in cultured mammalian and insect cells causes an accumulation of ER ceramides, leading to a structural collapse of ER exit sites and induction of mitochondrial apoptosis [18, 23]. These phenotypes are suppressed by blocking de novo ceramide synthesis, stimulating ER export of ceramides, or targeting SMS1 to the ER, supporting a role of SMSr as a critical regulator of ER ceramide levels. The physiological relevance of SMSr-mediated CPE production and the consequences of its acute disruption in mammals remain to be established
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