A Dictyostelium discoideum Mutant That Missorts and Oversecretes Lysosomal Enzyme Precursors Is Defective in Endocytosis

• Published in: The Journal of cell biology Vol. 109; no. 4; pp. 1445 - 1456
• Main Authors: Ebert, David L., Freeze, Hudson H., Richardson, Jan, Dimond, Randall L., Cardelli, James A.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01-10-1989; The Rockefeller University Press
• Subjects: alpha -mannosidase; beta -glucosidase; beta-Glucosidase - biosynthesis; beta-Glucosidase - genetics; Biochemistry. Physiology. Immunology. Molecular biology; Biological and medical sciences; Cell Fractionation; Cells; Centrifugation; Dictyostelium - enzymology; Dictyostelium - genetics; Dictyostelium - physiology; Endocytosis; Endosomes; Enzyme Precursors - biosynthesis; Enzyme Precursors - genetics; Enzymes; Fundamental and applied biological sciences. Psychology; Glucosidases - genetics; Kinetics; lysosomal enzymes; Lysosomes; Lysosomes - enzymology; Mannosidases - biosynthesis; Mannosidases - genetics; Mutation; Oligosaccharides; Oligosaccharides - isolation & purification; Oligosaccharides - metabolism; Protein Processing, Post-Translational; Protozoa; Receptors; Secretion; Stem cells; Protozoa; Fractionation; Radiolabelling; Gel electrophoresis; Enzyme; Secretion; Lysosome; Precursor; Strain; Sorting; Dictyostelium discoideum; Endocytosis; Immunoprecipitation reaction; β-D»-Glucosidase; Mutation; Defectivity; α-D-Mannosidase; Mycetozoa
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.109.4.1445

A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolable pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, α-mannosidase and β-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled α-mannosidase and 86% of its radiolabeled β-glucosidase as precursor polypeptides compared to the secretion of <10% of these forms from wild-type cells. Neither α-mannosidase nor β-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both α-mannosidase and β-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild-type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the α-mannosidase precursor by HMW570 was not accompanied by major alterations in N-linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3 H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3 H]dextran was predominately located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.