Identification of a distinct pool of sphingomyelin involved in the sphingomyelin cycle

Sphingomyelin (SM) is a membrane phosphosphingolipid that has recently been identified as a key component of the SM cycle. In this signal transduction pathway, extracellular inducers such as tumor necrosis factor alpha cause hydrolysis of membrane SM, resulting in the generation of the lipid second messenger ceramide. Only 10-20% of cellular SM appears to be involved in the SM cycle, raising the possibility of the existence of a unique "signaling" pool of SM. The existence and subcellular location of such a pool were investigated. Using bacterial sphingomyelinase from Staphylococcus aureus (bSMase), we first characterized two pools of SM, identified as an outer leaflet bSMase-sensitive pool and a distinct bSMase-resistant pool. These pools were further characterized by their differential solubility in Triton X-100 and by their kinetics of labeling. The signaling pool of SM was distinguished by the following: 1) resistance to bSMase, 2) solubility in Triton X-100, and 3) delayed labeling kinetics. In subfractionation studies, the signaling pool of SM co-fractionated with the plasma membrane. Since the SM cycle involves a cytosolic sphingomyelinase and the intracellular release of choline phosphate, this pool of SM appears to localize to the inner leaflet of the plasma membrane (or to a closely related compartment). These results identify a unique signaling pool of SM that undergoes significant hydrolysis (20-40%) in response to inducers of the SM cycle.