INTRACELLULAR DEGRADATION OF TYPE-I COLLAGEN AND FIBRONECTIN IN HUMANLUNG FIBROBLASTS - EVIDENCE AGAINST DEGRADATION IN PRE-LYSOSOMAL COMPARTMENTS

Fibroblasts degrade about 15% of newly synthesised collagen within the cell before it can be secreted. When the helical structure of collage n is disrupted, about 30% is degraded intracellularly. To determine if collagen degradation occurs in a pre-lysosomal compartment, the passa ge of type I collagen out of the endoplasmic reticulum or Golgi was in hibited by incubating human lung fibroblasts with brefeldin A or monen sin. In both cases, the type I collagen retained within the cell was s table over a 20 h period. Disrupting the helical structure of collagen with cis-hydroxyproline, 2,2'-bipyridyl or ethyl 3,4-dihydroxybenzoat e did not alter the stability of type I collagen in brefeldin or monen sin-treated cells. Incubating permeabilised cells in the presence of G TP gamma S (guanosine 5'-(3-O-thio)triphosphate), which blocks transpo rt out of the endoplasmic reticulum, also resulted in the stable reten tion of type I collagen. Addition of dithiothreitol to permeabilised c ells failed to initiate intracellular degradation. Similar results wer e obtained with fibronectin. Both normal fibronectin and fibronectin i n which canavanine replaced arginine were stable for 20 h in cells tre ated with brefeldin A or monensin. The degradation of native collagen is sensitive to inhibition by a cell-permeable cysteine proteinase inh ibitor (ALLN) but is insensitive to chloroquine (which raises the pH o f acidic intracellular compartments), whereas the degradation of abnor mal collagen was sensitive to both ALLN and chloroquine. These results argue against the intracellular degradation of collagen or fibronecti n in a pre-lysosomal compartment.