Abstract
We are investigating the molecular mechanisms involved in the localization of lysosomal enzymes in Dictyostelium discoideum, an organism that lacks any detectable mannose-6-phosphate receptors. The lysosomal enzymes alpha-mannosidase and beta-glucosidase are both initially synthesized as precursor polypeptides that are proteolytically processed to mature forms and deposited in lysosomes. Time course experiments revealed that 20 min into the chase period, the pulse-labeled alpha-mannosidase precursor (140 kD) begins to be processed, and 35 min into the chase 50% of the polypeptides are cleaved to mature 60 and 58-kD forms. In contrast, the pulse-labeled beta-glucosidase precursor (105 kD) begins to be processed 10 min into the chase period, and by 30 min of the chase all of the precursor has been converted into mature 100-kD subunits. Between 5 and 10% of both precursors escape processing and are rapidly secreted from cells. Endoglycosidase H treatment of immunopurified radioactively labeled alpha-mannosidase and beta-glucosidase precursor polypeptides demonstrated that the beta-glucosidase precursor becomes resistant to enzyme digestion 10 min sooner than the alpha-mannosidase precursor. Moreover, subcellular fractionation studies have revealed that 70-75% of the pulse-labeled beta-glucosidase molecules move from the rough endoplasmic reticulum (RER) to the Golgi complex less than 10 min into the chase. In contrast, 20 min of chase are required before 50% of the pulse-labeled alpha-mannosidase precursor exits the RER. The beta- glucosidase and alpha-mannosidase precursor polypeptides are both membrane associated along the entire transport pathway. After proteolytic cleavage, the mature forms of both enzymes are released into the lumen of lysosomes. These results suggest that beta- glucosidase is transported from the RER to the Golgi complex and ultimately lysosomes at a distinctly faster rate than the alpha- mannosidase precursor. Thus, our results are consistent with the presence of a receptor that recognizes the beta-glucosidase precursor more readily than the alpha-mannosidase precursor and therefore more quickly directs these polypeptides to the Golgi complex.
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Selected References
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- Cardelli J. A., Mierendorf R. C., Jr, Dimond R. L. Initial events involved in the synthesis of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum. Arch Biochem Biophys. 1986 Jan;244(1):338–345. doi: 10.1016/0003-9861(86)90122-0. [DOI] [PubMed] [Google Scholar]
- Cladaras M. H., Kaplan A. Maturation of alpha-mannosidase in Dictyostelium discoideum. Acquisition of endoglycosidase H resistance and sulfate. J Biol Chem. 1984 Nov 25;259(22):14165–14169. [PubMed] [Google Scholar]
- Fischer H. D., Creek K. E., Sly W. S. Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors. J Biol Chem. 1982 Sep 10;257(17):9938–9943. [PubMed] [Google Scholar]
- Fitting T., Kabat D. Evidence for a glycoprotein "signal" involved in transport between subcellular organelles. Two membrane glycoproteins encoded by murine leukemia virus reach the cell surface at different rates. J Biol Chem. 1982 Dec 10;257(23):14011–14017. [PubMed] [Google Scholar]
- Free S. J., Loomis W. F. Isolation of mutations in Dictyostelium discoideum affecting alpha-mannosidase. Biochimie. 1974;56(11-12):1525–1528. doi: 10.1016/s0300-9084(75)80276-8. [DOI] [PubMed] [Google Scholar]
- Freeze H. H., Yeh R., Miller A. L., Kornfeld S. Structural analysis of the asparagine-linked oligosaccharides from three lysosomal enzymes of Dictyostelium discoideum. Evidence for an unusual acid-stable phosphodiester. J Biol Chem. 1983 Dec 25;258(24):14874–14879. [PubMed] [Google Scholar]
- Hoflack B., Kornfeld S. Lysosomal enzyme binding to mouse P388D1 macrophage membranes lacking the 215-kDa mannose 6-phosphate receptor: evidence for the existence of a second mannose 6-phosphate receptor. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4428–4432. doi: 10.1073/pnas.82.13.4428. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lodish H. F., Kong N., Snider M., Strous G. J. Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature. 1983 Jul 7;304(5921):80–83. doi: 10.1038/304080a0. [DOI] [PubMed] [Google Scholar]
- Mierendorf R. C., Jr, Cardelli J. A., Dimond R. L. Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum. J Cell Biol. 1985 May;100(5):1777–1787. doi: 10.1083/jcb.100.5.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mierendorf R. C., Jr, Cardelli J. A., Livi G. P., Dimond R. L. Synthesis of related forms of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum. J Biol Chem. 1983 May 10;258(9):5878–5884. [PubMed] [Google Scholar]
- Owada M., Neufeld E. F. Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? Evidence from I-cell disease. Biochem Biophys Res Commun. 1982 Apr 14;105(3):814–820. doi: 10.1016/0006-291x(82)91042-7. [DOI] [PubMed] [Google Scholar]
- Paigen K. Acid hydrolases as models of genetic control. Annu Rev Genet. 1979;13:417–466. doi: 10.1146/annurev.ge.13.120179.002221. [DOI] [PubMed] [Google Scholar]
- Pannell R., Wood L., Kaplan A. Processing and secretion of alpha-mannosidase forms by Dictyostelium discoideum. J Biol Chem. 1982 Aug 25;257(16):9861–9865. [PubMed] [Google Scholar]
- Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sly W. S., Fischer H. D. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochem. 1982;18(1):67–85. doi: 10.1002/jcb.1982.240180107. [DOI] [PubMed] [Google Scholar]