Ceramide Accumulation Uncovers a Cycling Pathway for the cis-Golgi Network Marker, Infectious Bronchitis Virus M Protein

Maceyka, Michael; Machamer, Carolyn E.

Independent studies demonstrate that the lipid compositions of membranes differ at each stage of the secretory pathway (Keenan and Morre, 1970; Cluett et al., 1997; for review see van Meer, 1993) . Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), the precursor to all gangliosides, are one class of lipids thought to increase in relative concentration through the secretory pathway. Ceramide, the precursor of sphingolipids, is synthesized in the ER. In rat liver, ceramide is converted into the different classes of sphingolipids by enzymes localized to the CGN and the cis -and medial -Golgi cisternae (Futerman et al., 1990; Futerman and Pagano, 1991) . When expressed from a recombinant vaccinia virus, IBV M is localized to the CGN in several cell types, and its localization presumably overlaps with that of SM and GlcCer synthase activities. This led us to speculate that there may be a link between sphingolipid synthesis and the localization of IBV M. To address this question we tested the effects of three sphingolipid synthesis inhibitors on the steady-state localization of IBV M. We observed a dramatic redistribution of IBV M induced by one of these inhibitors, the glucosylceramide analogue d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Use of upstream inhibitors coupled with lipid analysis suggested that the PDMP effects are mediated by the accumulation of the precursor ceramide. Because IBV M can be induced to move to the ER, we propose that IBV M is at least in part localized by retrieval mechanisms.