A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis.

Abstract

Abstract. 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, o~-mannosidase and /3-glucosidase. A few hours into the chase period, HMW570 had secreted 95 % of its radiolabeled ot-mannosidase and 86% of its radiolabeled ~-glucosidase as precursor polypeptides compared to the secretion of <10% of these forms from wild-type cells. Neither a-mannosidase nor/$-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both a-mannosidase and/~-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 a-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 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.

Phototoxicity of halogenofluorescein derivatives in Dictyostelium discoideum amoebae: comparison of 2",4",5",7'-tetrabromofluorescein- and 4",5'-diiodofluorescein dextran

“…Kinetics of entry of Br,FD in Dictyostelium amoebae (Fig. 5 ) followed a course similar to that reported for F D or 1,FD (Ebert et al, 1989;Bof et al, 1992;Labrousse & Satre, 1993;Klein & Satre, 1986). Br,FD was internalized with an influx rate of 0.007 pl per cell min-' up to a final plateau corresponding to an apparent volume of 0-63 pl per cell.…”

“…In the slime mould Dictyostelium discoideum, endocytosis constitutes the major pathway for nutrient uptake in amoebae (North, 1983). This haploid organism is well adapted for genetic studies and mutants defective in phagocytosis and fluid-phase pinocytosis have already been obtained Waddell et al, 1987;Ebert et al, 1989;Bof et al, 1992;Labrousse & Satre, 1993). Clathrin-deficient amoebae, created by antisense RNA technique, are severely impaired in fluid-phase pinocytosis (O'Halloran & Anderson, 1992).…”

A PTEN-related 5-Phosphatidylinositol Phosphatase Localized in the Golgi

“…We therefore wondered whether PLIP is involved in the regulation of protein processing in the Golgi or transport from the Golgi to the lysosome. To examine this, we analyzed the maturation of ␣-mannosidase and ␤-glucosidase in PLIP null cells (47,48). ␣-Mannosidase is synthesized as a 140-kDa precursor and subsequently processed to an 80-kDa intermediate form and a mature 58-kDa subunit in the late Golgi or early endosome.…”

PsB Multiprotein Complex of

“…Generally, protein oligomerization occurs in the endoplasmic reticulum (Hurtley and Helenius, 1989), suggesting that one of the PsB complex subunits acts as an address tag for the entire complex. Indeed, it is possible that the oligosaccharide modifications of the PsB complex proteins are involved since glycosylation has been shown to be involved in intracellular targeting in Dictyostelium as well as other systems (Ebert et al, 1989;Fiedler and Simons, 1995;Freeze et al, 1989;Matter and Mellman, 1994). In addition, nothing is known about the biochemical make up of the PSVs and especially about the PSV membrane which must be involved in the import of the protein cargo, the receipt of the signal for secretion and the eventual fusion of the PSVs with the plasma membrane during secretion.…”