Effect of Tunicamycin on Transport of Lysosomal Enzymes in Cultured Skin Fibroblasts

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Abstract
After an initial lag phase, tunicamycin (0.12–3.6 μM) inhibits the secretion of β‐N‐acetylglucosaminidase by human skin fibroblasts. The inhibition persists for up to 30 h. Thereafter a more than fivefold‐increased rate of secretion is observed for at least 4 days. The intracellular β‐N‐acetylglucos aminidase activity decreases within 9 days in the presence of 0. 36 μM tunicamycin to 55% of that of controls. β‐N‐Acetylglucosaminidase formed in the presence of tunicamycin is only poorly recognized by fibroblasts, suggesting that tunicamycin interferes with the formation of the phosphomannosyl residues that function on β‐N‐acetylglucosaminidase as a recognition marker for the cell surface receptors present on fibroblasts. Tunicamycin (0.36–3.6 μM) inhibits endocytosis of exogenous lysosomal enzymes only after a preincubation of 24 h in the presence of the drug. Control experiments indicated that this inhibition is not due to a general inhibition of receptor‐mediated endocytosis. Incubation with tunicamycin leads furthermore to a loss of cell‐surface‐associated lysosomal enzymes.These findings indicate that tunicamycin interferes with the expression of phosphomannose receptors on the cell surface of fibroblasts and the synthesis of the phosphomannosyl residues on lysosomal enzymes after an initial lag phase. These alterations are associated with an increased accumulation of β‐N‐acetylglucosaminidase extracellularly, a loss of that enzyme from intracellular sites and from the cell surface. A similar mislocation of lysosomal enzymes in mucolipidosis II fibroblasts that is associated with a deficiency of the recognition marker on lysosomal enzymes but normal expression of the phosphomannose receptors led to the hypothesis that lysosomal enzymes undergo a secretion‐endocytosis process during their transfer into lysosomes [Hickman, S. & Neufeld, E. F. (1972) Biochem. Biophys. Res. Commun. 49, 992–999]. The present results suggest that phosphomannosyl residues within N‐glycosidically linked oligosaccharide chains on lysosomal enzymes [von Figura, K. & Klein, U. (1979) Eur. J. Biochem. 94. 347–354] function either intracellularly or on the cell surface as signal for their transfer into lysosomes.