Initial events involved in the synthesis of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum.
In Dictyostelium discoideum the lysosomal enzyme alpha-mannosidase is initially synthesized in vivo as a 140,000 Mr protein which is subsequently processed into two mature acidic glycoproteins of 60,000 and 58,000 Mr. To investigate the initial events involved in the synthesis of this protein, mRNA isolated from growing cells was translated in vitro and the resulting protein products were immunoprecipitated with antibodies prepared against the purified enzyme. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of an immunoprecipitable 120K protein that was identified as the alpha-mannosidase primary translation product by a variety of criteria. Translation in vitro in the presence of dog pancreas microsomes resulted in the conversion of the 120K primary translation product to a 140K form. This 140K species was not accessible to added trypsin under conditions preserving membrane integrity, suggesting it is sequestered in the lumen of the endoplasmic reticulum following synthesis. Treatment of either the in vitro modified or cellular 140K alpha-mannosidase precursors with endoglycosidase H resulted in the appearance of proteins 2K larger than the primary translation product. The pulse-labeled cellular precursor and the in vitro processed form have similar isoelectric points as revealed by two-dimensional gel electrophoresis. These results imply that the precursor is N-glycosylated in the endoplasmic reticulum possibly without removal of the signal sequence and that the majority of acidic modifications are added late in the post-translational pathway.
D000818:Animals; D001665:Binding Sites; D002474:Cell-Free System; D004023:Dictyostelium / Q000201:enzymology*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000502:physiology; D012333:RNA, Messenger / Q000502:physiology; D011817:Rabbits; D043323:alpha-Mannosidase
D012331:RNA, Fungal; D012333:RNA, Messenger; D008361:Mannosidases; D043323:alpha-Mannosidase