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

Maceyka, Michael; Machamer, Carolyn E.

It is not yet clear how ceramide induces the changes in anterograde traffic and protein localization. Recent studies have shown that ceramide (Hannun, 1994) and its metabolites (Hakomori, 1990 ) play a major role as second messengers. Preliminary experiments with both the ceramidase inhibitor N-oleoylethanolamine and exogenously added sphingosine suggest that the ceramide breakdown product sphingosine does not play a role in the localization of IBV M (Maceyka, M., and C. Machamer, unpublished observations). Ceramide activates a cytosolic protein phosphatase (Dobrowsky and Hannun, 1992; Wolff et al., 1994) that can be inhibited by okadaic acid. Preliminary experiments suggest that okadaic acid does not block the PDMPinduced changes (Maceyka, M., and C. Machamer, unpublished observations). Ceramide could be activating a protein kinase (Mathias et al., 1991) , but this kinase has not been fully characterized. Another possibility is that increased ceramide within the lipid bilayer directly affects the localization of IBV M. Earlier work from our laboratory showed that the first transmembrane domain of IBV M can target chimeras to the CGN (Swift and Machamer, 1991; Machamer et al., 1993) . It is possible that CGN bilayers have distinct lipid domains, and that these domains contain different sets of proteins, analogous to glycosphingolipid rafts. Segregation of membrane proteins involved in vesicular traffic, such as SNAREs, would lead to mobile and immobile domains. We hypothesize that under normal conditions, IBV M would be targeted to an immobile domain and escaped molecules would be cycled back through the ER. Elevated levels of ceramide could disrupt these immobile lipid domains, inducing IBV M to cycle. Alternatively, perhaps ceramide binds to IBV M transmembrane domains, preventing an interaction with immobile lipid domains.