Brefeldin A inhibits degradation as well as production and secretion of collagen in human lung fibroblasts

Brefeldin A (BFA) inhibits protein secretion, collapses the Golgi complex into the endoplasmic reticulum (ER), causes redistribution of processing enzymes normally resident in the Golgi to the ER, and uncouples the proximal and distal regions of the secretory pathway. We used BFA to determine where intracellular degradation of newly synthesized collagen degradation occurs. In normal human fetal lung fibroblasts, BFA (50 ng/ml) completely blocked collagen secretion and reduced collagen production by two-thirds. In cells synthesizing collagen under normal conditions, intracellular degradation was about 16%; BFA (50 ng/ml) reduced degradation to less than 5%. In cells induced to synthesize structurally abnormal collagen (by incubation with the proline analog cis-hydroxyproline), degradation was approximately 33%; BFA reduced this level to less than 10%. When the y axes were scaled appropriately, the dose-response curves for collagen degradation +/- cis-hydroxyproline versus BFA concentration coincided. A pulse-chase experiment demonstrated that BFA did not inhibit hydroxylation of prolyl residues, a major posttranslational modification of collagen that occurs in the ER, and that inhibition of degradation was independent of inhibition of collagen synthesis. Immunofluorescence examination revealed that BFA redistributed Golgi glycoproteins to the ER. At the ultrastructural level, Golgi complex could not be found in fibroblasts exposed to BFA for 1 h; however, clusters of small vesicles were observed. A different structure, comprising one or two lamellae and resembling a partial Golgi complex, was observed in cells incubated with BFA for 6 h. This structure was adjacent to ER but far from the nucleus. In addition, the ER was devoid of ribosomes. The inhibition of intracellular collagen degradation by BFA indicates that collagen degradation does not occur in the ER. Rather, it suggests that collagen degradation occurs beyond the BFA block, perhaps in the trans-Golgi network.