Lysosomal Enzymes in Dictyostelium discoideum Are Transported to Lysosomes at Distinctly Different Rates

We are investigating the molecular mechanisms involved in the localization of lysosomal enzymes in Dictyostelium discoideum, an organism that lacks any detectable mannose-6-phosphate receptors. The lysosomal enzymes α-mannosidase and β-glucosidase are both initially synthesized as precursor polypeptides that are proteolytically processed to mature forms and deposited in lysosomes. Time course experiments revealed that 20 min into the chase period, the pulse-labeled α-mannosidase precursor (140 kD) begins to be processed, and 35 min into the chase 50% of the polypeptides are cleaved to mature 60 and 58-kD forms. In contrast, the pulse-labeled β-glucosidase precursor (105 kD) begins to be processed 10 min into the chase period, and by 30 min of the chase all of the precursor has been converted into mature 100-kD subunits. Between 5 and 10% of both precursors escape processing and are rapidly secreted from cells. Endoglycosidase H treatment of immunopurified radioactively labeled α-mannosidase and β-glucosidase precursor polypeptides demonstrated that the β-glucosidase precursor becomes resistant to enzyme digestion 10 min sooner than the α-mannosidase precursor. Moreover, subcellular fractionation studies have revealed that 70-75% of the pulse-labeled β-glucosidase molecules move from the rough endoplasmic reticulum (RER) to the Golgi complex less than 10 min into the chase. In contrast, 20 min of chase are required before 50% of the pulse-labeled α-mannosidase precursor exits the RER. The β-glucosidase and α-mannosidase precursor polypeptides are both membrane associated along the entire transport pathway. After proteolytic cleavage, the mature forms of both enzymes are released into the lumen of lysosomes. These results suggest that β-glucosidase is transported from the RER to the Golgi complex and ultimately lysosomes at a distinctly faster rate than the α-mannosidase precursor. Thus, our results are consistent with the presence of a receptor that recognizes the β-glucosidase precursor more readily than the α-mannosidase precursor and therefore more quickly directs these polypeptides to the Golgi complex.