CHARACTERIZATION, SUBCELLULAR-LOCALIZATION, AND DEVELOPMENTAL REGULATION OF A CYSTEINE PROTEINASE FROM DICTYOSTELIUM-DISCOIDEUM

Previous studies showed that vegetative cells of Dictyostelium discoid eum make a cysteine proteinase called proteinase-1, which contains mul tiple residues of GlcNAc-1-P linked directly to peptidyl serines. As a prelude to understanding the function of this novel carbohydrate modi fication, we purified and extensively characterized this proteinase in terms of its enzymatic activity, subcellular localization, and develo pmental regulation, The purified enzyme has an apparent molecular weig ht of 38 kDa in heat-denatured, reducing SDS/PAGE and 55 kDa under non reducing conditions. Native gel electrophoresis and isoelectric focusi ng revealed two protein bands with equal activity and having pI values of 2.5 and 2.6. Even more complex patterns are found in non-heat-dena tured SDS/PAGE gels, However, partial amino acid sequencing of the pur ified protein gave predominantly a single sequence. The enzyme is inhi bited by trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, Na-p- tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chlorometh yl ketone, and leupeptin, has a pH optimum of 5.0, and cofractionates with lysosomal enzymes in bacterially grown cells. It appears to compr ise about 90% of the total cysteine proteinase activity in cells at a time when the cells have just finished clearing the bacterial lawn, Pr ior to this point and after the onset of development, its level is 2- to 20-fold lower. This remarkably fine regulation parallels the develo pmental regulation of other cysteine proteinases in Dictyostelium. Bas ed on these results it appears that proteinase-1 may be primarily used for specialized proteolysis just before the onset of development rath er than for simply digesting the bacteria for food. (C) 1995 Academic Press, Inc.