Brefeldin A inhibits the endocytosis of plasma-membrane-associated heparan sulphate proteoglycans of cultured rat ovarian granulosa cells.

ABSTRACT

Rat ovarian granulosa cells were labelled with [35S]sulphate for 0.5-20 h and chased in the presence or absence of 1-2 micrograms/ml of brefeldin A (BFA) for up to 21 h. Heparan [35S]sulphate (HS) proteoglycans from the culture medium, plasma membrane and intracellular fractions were then analysed by gel chromatography. In the absence of BFA, about 85% of the plasma membrane-associated HS proteoglycans were endocytosed and subsequently degraded intracellularly. Recirculation of the HS proteoglycans between the intracellular pool and the cell surface was not observed. Exposing the cells to BFA for less than 1 h did not influence the turnover of the HS proteoglycans, whereas the effect of the drug on the Golgi functions reached a maximum in approx. 10 min. When the cells were treated with BFA for more than 1-2 h, the rate of endocytosis of HS proteoglycans was reduced to about 50% of the control. The delivery of endocytosed HS proteoglycans to lysosomes were not affected by the drug. Cycloheximide also reduced the endocytosis of HS proteoglycans, but not as much as BFA, indicating that the inhibitory effect of BFA can be only partly accounted for by a block of protein transport from the endoplasmic reticulum to the plasma membrane. In contrast with the endocytosis of HS proteoglycans, neither that of 125I-transferrin, known to be mediated by clathrin-coated vesicles, nor that of 125I-ricin, a marker molecule for bulk endocytosis, was affected by BFA. The half-life of 125I-transferrin and 125I-ricin in the plasma membrane was about 10 and 25 min respectively compared with about 5 h for the HS proteoglycans. Altogether, these results indicate that the endocytosis of plasma-membrane-associated HS proteoglycans is mediated by different mechanisms than the endocytosis of most other cell-surface proteins. Further, the mechanisms involved in the endocytosis of HS proteoglycans are sensitive to BFA.