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 α 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.