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

Maceyka, Michael; Machamer, Carolyn E.

To express the IBV M protein, BHK-21 cells were infected with a recombinant vaccinia virus encoding IBV M (Machamer and Rose, 1987) . 4 h after infection the cells were treated with cycloheximide for 1 h to chase newly synthesized M protein out of the ER. Sphingolipid synthesis inhibitors were then added and the cells incubated for another 1 h before processing for immunofluorescence ( Fig. 2) . In control cells, IBV M exhibited a tight, juxtanuclear staining pattern that colocalized with Golgi markers. The Golgi localization pattern of IBV M was unchanged by treatment with either ␤ CA or FB1, suggesting that ongoing sphingolipid synthesis is not required for proper localization of IBV M. In contrast, PDMP-treated cells showed a marked change in the staining pattern of the IBV M pro-tein after 1 h. The localization of IBV M in the presence of PDMP changed from Golgi to ER based on the absence of strong juxtanuclear staining and the presence of nuclear rim staining and a tubulo-reticular staining pattern that colocalized with ER markers. Treatment of infected cells with lower concentrations of PDMP had no effect on IBV M localization and very little effect on SM synthesis (data not shown). We next tested the kinetics of PDMP-induced mislocalization of IBV M. Cells were infected as before and treated for 1 h with cycloheximide. Then cells were treated with PDMP for varying lengths of time up to 1 h (Fig. 3) . Redistribution was first observed at 15 min and became maximal at 60 min. This redistribution was rapidly reversible, such that 30 min after washing out PDMP, IBV M had moved back to the Golgi region.