CERAMIDE ACCUMULATION UNCOVERS A CYCLING PATHWAY FOR THE CIS-GOLGI NETWORK MARKER, INFECTIOUS-BRONCHITIS VIRUS M-PROTEIN

The M glycoprotein from the avian coronavirus, infectious bronchitis v irus (IBV), contains information for localization to the cis-Golgi net work in its first transmembrane domain. We hypothesize that localizati on to the Golgi complex may depend in part on specific interactions be tween protein transmembrane domains and membrane lipids, Because the s ite of sphingolipid synthesis overlaps the localization of IBV M, we a sked whether perturbation of sphingolipids affected localization of IB V M, Short-term treatment with two inhibitors of sphingolipid synthesi s had no effect on localization of IBV M or other Golgi markers, Thus, ongoing synthesis of these lipids was not required for proper localiz ation. Surprisingly, a third inhibitor, o-1-phenyl-2-decanoylamino-3-m orpholino-1-propanol (PDMP)I shifted the steady-state distribution of IBV M from the Golgi complex to the ER, This effect was rapid and reve rsible and was also observed for ERGIC-53 but not for Golgi stack prot eins, At the concentration of PDMP used, conversion of ceramide into b oth glucosylceramide and sphingomyelin was inhibited. Pretreatment wit h upstream inhibitors partially reversed the effects of PDMP, suggesti ng that ceramide accumulation mediates the PDMP-induced alterations. I ndeed, an increase in cellular ceramide was measured in PDMP-treated c ells. We propose that IBV M is at least in part localized by retrieval mechanisms. Further, ceramide accumulation reveals this cycle by upse tting the balance of anterograde and retrograde traffic and/or disrupt ing retention by altering bilayer dynamics.