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

Maceyka, Michael; Machamer, Carolyn E.

Interestingly, it has been shown that myriocin, an inhibitor of sphingolipid synthesis thought to inhibit the same step as ␤CA, reduces the rate of transport of a glycosylphosphatidylinositol-linked protein but not other proteins out of the ER in yeast (Horvath et al., 1994) . It would be interesting to test whether inhibition of ceramide synthesis in mammalian cells also slows the rate of transport of glycosylphosphatidylinositol-linked proteins in mammalian cells. However, neither ␤CA nor FB1 had an effect on the rate of anterograde traffic of VSV G or the localization of IC, CGN, or Golgi stack proteins. When we quantified the effects of these two inhibitors on the immunofluorescence Figure 9 . Exogenous C 6 Cer did not redistribute IBV M or ER-GIC-53. BHK cells (IBV M, Man II, and ␤-COP) or Vero cells (ERGIC-53) were infected with a recombinant vaccinia virus expressing IBV M. 4 h after infection the cells were treated with 50 g/ml cycloheximide. At 5 h after infection cells were incubated for 1 h in serum-free DME with 0.34 mg/ml defatted BSA with or without 25 M C 6 Cer. Cells were then prepared for indirect immunofluorescence and stained with the indicated antibodies. For each experimental series, the Nomarski image is shown on the left and the fluorescence image on the right. Bar, 10 m. Figure 10 . Current models for how IBV M maintains its steady-state distribution and how PDMP might alter this distribution. (A) Evidence presented here shows that IBV M can be induced to redistribute to the ER, indicating that IBV M has information necessary for traffic to the ER. We propose that IBV M normally maintains its steady-state distribution by cycling through the ER at some basal rate. (B) If IBV M is cycling through the ER normally, then its localization would depend on the balance of anterograde and retrograde traffic rates. As PDMP slows anterograde traffic, IBV M could accumulate in the ER when the balance of membrane traffic is disrupted (left arrow). Alternatively, PDMP may act by disrupting the retention of IBV M, causing it to move out of the Golgi (right arrow). It is also possible that redistribution of IBV M requires both effects of PDMP. staining pattern of IBV M, we did detect a slight shift toward the intermediate staining pattern (Fig. 7) . Whether this represents an altered distribution pattern or subtle effects on Golgi structure caused by these inhibitors is unclear. From these results we conclude that ongoing sphingolipid synthesis is not required for either normal rates of anterograde traffic or proper localization of Golgi proteins. However, the glucosylceramide analogue PDMP, at a concentration that inhibits both GlcCer and SM synthases, causes a redistribution of IBV M to the ER and a slowing of anterograde traffic in BHK-21 cells. Why does PDMP have these effects while ␤CA and FB1 do not? It is likely that PDMP exerts its effects by causing the accumulation of ceramide. Other groups have found that the various effects of PDMP and its active analogues were concomitant with (Uemura et al., 1990; Shayman et al.; Rani et al., 1995) or actually caused by increases in ceramide concentrations (Abe et al., 1996; Posse de Chaves et al., 1997) . In BHK-21 cells treated with 100 M PDMP for 1 h we measured an increase in newly synthesized ceramide (Fig. 8 A) , showing at least a correlation between levels of newly synthesized ceramide and the effects of PDMP on protein traffic and localization. The fact that pretreatment wi