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

Maceyka, Michael; Machamer, Carolyn E.

We then looked at a protein which cycles through the cis -Golgi. As we did not have access to antibodies that crossreact with known markers in BHK-21 cells, we examined ERGIC-53 in Vero cells. IBV M localizes to the CGN in these cells and is also redistributed to the ER by PDMP (data not shown). ERGIC-53 is an IC protein that cycles between the ER, IC, and Golgi stack (Lippincott-Schwartz et al., 1990; Schindler et al., 1993) . Vero cells were treated with 50 g/ml cycloheximide for 1 h. PDMP (100 M) was added, and dishes were fixed at the indicated times and prepared for indirect immunofluorescence. After 1 h in PDMP, the remaining dishes were washed three times with 1 ml DME with serum and incubated with cycloheximide-containing medium for the indicated time before being prepared for indirect immunofluorescence. For each experimental series, the Nomarski image is shown on the left and the fluorescence image on the right. Bar, 10 m. with cycloheximide for 1 h and then with or without 100 M PDMP for an additional hour before being fixed and prepared for immunofluorescence (Fig. 5) . Similar to the effects on IBV M, PDMP treatment shifted the steady-state distribution of ERGIC-53 to the ER. In addition, neither ␤ CA nor FB1 had any effect on the localization of ER-GIC-53 (data not shown).