Intracellular reduction of disulfide bonds in endocytosed macromolecules
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Abstract
It was known that Chinese hamster ovary cells cleaved the disulfide bonds of endocytosed 125I-tyramine-SS-poly(D-lysine) (125I-tyn-SS-PDL) 1 to 3 h after uptake, in an organelle present in the light density fraction of a 17% Percoll gradient, a complex fraction which contains the Golgi apparatus and the trans-Golgi network (TGN), the endoplasmic reticulum, plasma membrane fragments and endosomes. A discontinuous sucrose gradient was used to isolate a pure Golgi/TGN fraction, and cell pretreatment with Brefeldin A (BFA) was used to separate Golgi from TGN. Pulse chase experiments showed that TGN was the only cellular fraction in which significant cleavage of 125I-tyn-SS-PDL occurred during the 1 to 3 h post-labeling period. 125I-PDL used as a control, reached the TGN but was not degraded. BFA (1.0 microgram/ml) caused disassembly of the Golgi, and led to a redistribution of the Golgi marker galactosyl transferase to the endoplasmic reticulum (ER), but caused only little change in the distribution of sialyltransferase, an enzyme found predominantly in the TGN. BFA did not influence the distribution of 125r-PDL in either the Golgi or the TGN fractions of pulse-labelled cells, indicating that PDL is endocytosed to the TGN but not to the Golgi stacks. BFA did not alter the kinetics or extent of intracellular reduction of 125I-tyn-SS-PDL occurring 1 to 3 h post-labeling, as measured either in intact cells or in the isolated TGN fraction. Since other studies provided strong evidence that the reduction of membrane-bound 125I-tyn-SS-PDL is catalyzed by membrane associated protein disulfide isomerase (PDI), the TGN fraction was tested for and found to contain PDI activity. The fraction had the capacity to reductively cleave 125I-tyn-SS-PDL added in vitro. The presence of PDI in the TGN was demonstrated both enzymatically and by immunoblotting, using a monoclonal anti-PDI antibody. All available data thus indicate that reduction of endocytosed disulfides occurs in the TGN, and is catalyzed by protein disulfide isomerase.
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Dissertation (Ph.D.)--Boston University Page 107 is missing in all copies
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