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

Maceyka, Michael; Machamer, Carolyn E.

The above immunofluorescence results suggested that PDMP might generally alter the distribution of proteins that have a dynamic localization mechanism. Interestingly, it has been shown that PDMP slows the rate of both anterograde vesicular traffic (Rosenwald et al., 1992) and endocytosis (Chen et al., 1995) in CHO cells. We tested whether PDMP also slowed anterograde traffic in BHK-21 cells. We used a plasma membrane protein, the well-characterized G protein from VSV. A pulse-chase experiment was performed either in the presence or absence of PDMP, ␤ CA, or FB1. The rate of accumulation of endo H-resistant VSV G was used as an assay for the rate of arrival at the medial -Golgi. Neither ␤ CA nor FB1 had any effect on the rate of anterograde traffic. However, PDMP increased the halftime for transport by ‫ف‬ 2.5-fold from 18 to 44 min (Fig. 6 ). Fig. 6 shows that neither ␤ CA nor FB1 had any effect on the transit rate of VSV G, suggesting that the decreased rate with PDMP was not due to a block in the ongoing synthesis of sphingolipids. PDMP inhibits the conversion of ceramide into glycosphingolipids and sphingomyelin. One possible explanation for the effects observed with PDMP was an accumulation of the precursor ceramide. ␤ CA and FB1 act upstream of PDMP (Fig. 1) , and the intermediates in sphingolipid synthesis between these steps and ceramide production are thought to be short lived (Merrill and Wang, 1986; Medlock and Merril, 1988) . To ask if ceramide accumulation might mediate the PDMP-induced slowing of anterograde traffic, cells were treated with ␤ CA or FB1 before PDMP. We performed a pulse-chase labeling experiment with cells pretreated with either ␤ CA or FB1 for 1 h before being chased in the presence or absence of ␤ CA or FB1 and PDMP (Fig. 6) . Pretreatment with the earlier inhibitors reduced the PDMP-induced slowing of anterograde traffic by about half, suggesting that accumulation of newly synthesized ceramide at least partially mediates this effect.