Anterograde and retrograde transport of neutral sphingomyelinase-2 between the Golgi and the plasma membrane

The activation of neutral sphingomyelinase-2 (nSMase2) and consequent ceramide production are implicated in many stress-induced signaling pathways. Trafficking of nSMase2 from the Golgi compartment to the plasma membrane (PM) in response to signaling stimuli has been described. However, the precise mechanisms of transport remain unknown. This study aimed to investigate the trafficking of nSMase2 between the Golgi and the PM. We show here that V5-nSMase2 localizes at the PM and Golgi in MCF-7 cells and confirm relocalization of nSMase2 to the PM at confluence. Although cycloheximide (CHX) treatment partially inhibited the Golgi localization of GFP-nSMase2, recovery of GFP-nSMase2 to an intracellular compartment was still observed after photobleaching. Moreover, in the presence of CHX, GFP- and V5-nSMase2 co-localized with endosomal/recycling markers. In HEK293 cells, activation of either protein kinase C-alpha or betaII, with the phorbol ester PMA led to relocalization of both wild-type and inactive nSMase2 to the pericentrion, a PKC-dependent subset of recycling endosomes. Finally, inhibition of nSMase2 endocytosis by K. +. depletion reduced the intracellular pool of nSMase2 and increased nSMase2 activity resulting in elevated ceramide levels. Altogether, these results suggest that nSMase2 traffics from the Golgi to the PM as a membrane protein en route to the cell surface and recycles back to the Golgi through the endosomal/recycling compartment. Moreover, the recycling of nSMase2 from the PM is important for its catalytic regulation.