A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis.
A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha-mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-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 alpha-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, [3H]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 [3H]dextran was predominantly 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.
Ebert
D L
DL
Department of Bacteriology, University of Wisconsin, Madison 53706.
Freeze
H H
HH
Richardson
J
J
Dimond
R L
RL
Cardelli
J A
JA
D002458:Cell Fractionation; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D004705:Endocytosis; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics*; D005959:Glucosidases / Q000235:genetics*; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D043323:alpha-Mannosidase; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics*
D004792:Enzyme Precursors; D009844:Oligosaccharides; D005959:Glucosidases; D008361:Mannosidases; D001617:beta-Glucosidase; D043323:alpha-Mannosidase