DIFFERENTIAL EFFECT OF BREFELDIN-A ON THE BIOSYNTHESIS OF HEPARAN-SULFATE AND CHONDROITIN DERMATAN SULFATE PROTEOGLYCANS IN RAT OVARIAN GRANULOSA-CELLS IN CULTURE

The subcellular localization of the enzymes involved in the glycosylat ion of proteoglycans was studied in rat ovarian granulosa cells by int erfering with the normal traffic in the Golgi apparatus using brefeldi n A. Cell cultures were metabolically labeled with S-35! sulfate and H-3!glucosamine, and the radiolabeled macromolecules were analyzed by ion-exchange and gel chromatography in combination with chondroitinas e or heparitinase treatment. In the absence of brefeldin A, the cells synthesized both dermatan sulfate proteoglycans (DSPGs) and heparan su lfate proteoglycans (HSPGs) which were isolated from the culture mediu m, the plasma membrane, and intracellular compartments. However, in th e presence of brefeldin A, the synthesized proteoglycans were almost e xclusively HSPGs and were found only in the intracellular compartment. Analyses of HSPGs synthesized in the presence of brefeldin A indicate d that: (i) the HS chains are synthesized on the same core protein as for the normal HSPGs; (ii) the chains are two to three times the norma l molecular size; and (iii) a significant proportion of the HS chains are normally sulfated. Brefeldin A induces a disassembly of the proxim al part of the Golgi complex, resulting in a redistribution of cis-, m edial-, and trans-Golgi resident enzymes back to the endoplasmic retic ulum (ER), and blocks the transport of proteins to the trans-Golgi net work. Our results indicate that the complete set of enzymes involved i n the biosynthesis of HS chains are localized in the ER/proximal part of the Golgi complex, whereas the enzymes involved in the elongation/s ulfation of DS chains are exclusively located in the trans-Golgi netwo rk. Furthermore, our results indicate that the enzymes involved in the biosynthesis of HS chains are specific to HS core proteins, since no DS core proteins were substituted with HS chains in the presence of br efeldin A.