Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: Evidence for a maturation model for the formation of lysosomes
The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11°C; acidification was limited to pH 6–6.5 at temperatures between 13°C and 17°C. In contrast, a rapid drop to pH 6–6.5 followed by acidification to pH 5–5.5 was observed at temperatures above 19°C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98:1757–1762). The timing of exposure of material internalized by fluid‐phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37°C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13° and 21°C, the rate of hydrolysis was reduced to 31–44% of that at 37°C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20°C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low‐density organelle containing lysosomal enzymes. Copyright © 1987 Wiley‐Liss, Inc.
Journal of Cellular Physiology
Digital Object Identifier (DOI)
Roederer, Mario; Bowser, Robert; and Murphy, Robert F., "Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: Evidence for a maturation model for the formation of lysosomes" (1987). Neurobiology. 643.