Details

Autor(en) / Beteiligte

• Ebert, David L.
• Freeze, Hudson H.
• Richardson, Jan
• Dimond, Randall L.
• Cardelli, James A.

Titel

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

Ist Teil von

• The Journal of cell biology, 1989-10, Vol.109 (4), p.1445-1456

Ort / Verlag

New York, NY: Rockefeller University Press

Erscheinungsjahr

1989

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.