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

Maceyka, Michael; Machamer, Carolyn E.

The pretreatment experiments as well as lipid analysis implicated ceramide as the mediator of PDMP effects. If this were true, addition of a soluble, short-chain analogue of ceramide might mimic the effects of PDMP. Indeed, one such cell-permeable analogue of ceramide, N -hexanoylsphingosine (C 6 Cer), reproduces the effects of PDMP on anterograde traffic of VSV G in CHO cells (Rosenwald and Pagano, 1993) . We first asked whether C 6 Cer slowed anterograde traffic in BHK-21 cells. A pulse-chase labeling experiment with VSV-infected cells was performed, with cells chased in the absence or presence of 25 M C 6 Cer. C 6 Cer slowed the rate of anterograde traffic in BHK-21 cells ‫-5.2ف‬fold (data not shown). We went on to use C 6 Cer in our indirect immunofluorescence assay. As expected, C 6 Cer had no effect on the localization of either Man II or ␤-COP (Fig. 9) . However, contrary to our expectation, C 6 Cer did not alter the localization of either IBV M or ER-GIC-53. This suggests that exogenously added C 6 Cer and ceramide generated by treatment with PDMP do not have the same effects. Two other soluble analogues of ceramide, C 2 Cer and C 8 Cer, were also tested and found to have no effect on the localization of IBV M (data not shown).