The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors.
The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006600:Hexosephosphates / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008360:Mannosephosphates / Q000378:metabolism*; D009154:Mutation; D010053:Ovary; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*
D006600:Hexosephosphates; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D006026:Glycoside Hydrolases