Dictyostelium discoideum spore germination: increases in proteinase activity are not directly coupled to the emergence of myxamoebae

Abstract

The accumulation of proteinase activity during the germination of Dictyostelium discoideum spores was examined under conditions in which the timing of germination events was varied. Spores had a single major proteinase, the aspartic proteinase ddAP58 (proteinase E), while the extracellular matrix which surrounds the spores in the fruiting body contained a number of lower-Mr cysteine proteinases, the most active being ddCP18. Very little peptide-nitroanilide-hydrolysing activity was detectable in spores, although some was associated with the matrix. During spore germination there were large increases in activity towards N-carbobenzoxy-L-tyrosyl-L-lysyl-Larginine 4-nitroanilide and N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide. The enzymes responsible for these activities (referred to as ZYKRase and BzPFRase respectively) were not identical as BzPFRase was much more sensitive to the cysteine proteinase inhibitor E-64 than was ZYKRase. When spores were heatactivated, the increases in activity coincided with the emergence of myxamoebae and the appearance of cysteine proteinases detected using electrophoresis in gelatin gels. For autoactivated spores, emergence was delayed by 0.5 to 1 h and proteinase accumulation lagged slightly behind emergence. If the spores were activated with DMSO, or if heat-activated spores were treated with sucrose after swelling, proteinase accumulation proceeded more rapidly than emergence. Thus temporal control of the accumulation of proteinases during germination varies according to the conditions used. In heat-activated spores, the timing of the increase was similar to that observed previously for P-glucosidase and trehalase, but the temporal controls were not the same as those for the latter enzymes when the other activation conditions were used. The results show that proteinase accumulation is not directly coupled to emergence but could be more closely linked to the late swelling stage which precedes emergence.

Formation of the Dictyostelium spore coat

“…It is not known whether the absence of two acid hydrolases (including N-acetyl-f3-D-glucosaminidase) from the interspore matrix is because they are not secreted or because they are incorporated into the spore coat. In addition, two GAS (-XXX and -XXXII), which are each associated with multiple secretory glycoproteins, accumulate in the interspore matrix but not the spore coat (West and Erdos, 1988); a similar observation has also been made for a protease using a different method (North et al, 1990). These GAS do not normally accumulate in the PSV.…”

Patterning of development in Dictyostelium discoideum: factors regulating growth, differentiation, spore dormancy, and germination

“…This same acid shocking technique has also revealed the presence of the two cysteine proteases in dormant spore extracts ). Using conventional SDS-PAGE techniques, these two protease activities normally have been shown to appear during spore germination (North et al 1990a). The results suggest packaging of the two proteases in early prespore cells and their gradual deactivation during slug migration and culmination; the acid shock treatment revealing cryptic protease activity may mimic lysosomal acidification by acidosomes (see Padh et al 1989Padh et al , 1991 during spore germination.…”

“…We also suspect that the germination cascade leads to the activation of acidosomes (see Padh et al 1991), which then fuse with the lysosomal/endosomal vacuolar system and activate resident lysosomal enzymes such as trehalase-1 and the developmental CP48 and CP43 proteases (Jackson et al 1982;Klein et al 1990;Gupta and Cotter 1990;North et al 1990a;North and Cotter 1991~). Presumably, this event precedes the spore-swelling stage, the hydrolysis of trehalose, and the utilization of the excess amino acid reserves including the large pools of Q, E, N, etc.…”

Acid-activatable Cysteine Proteinases in the Cellular Slime Mold

“…Net cysteine proteinase activity is high in vegetative cells but decreases during the developmental phase (3), partly as a result of secretion (3,4). Consequently, both the stalk cells and the dormant spores that are formed during development possess very low levels of detectable activity; however, some cysteine proteinase activity is found in the extracellular matrix surrounding spores in fruiting bodies (5). Net intracellular cysteine proteinase activity returns to appreciable levels during spore germination (5).…”