Sulfation in the Golgi lumen of Madin-Darby canine kidney cells is inhibited by brefeldin A and depends on a factor present in the cytoplasm and on Golgi membranes.
Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2-5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15-30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm.
Department of Biochemistry and Institute for Nutrition Research, University of Oslo, Oslo 0316, Norway.
Katja
K
Fjeldstad
Mona E
ME
Pedersen
Tram Thu
TT
Vuong
Svein Olav
SO
Kolset
Line Mari
LM
Nordstrand
Kristian
K
Prydz
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology*; D002460:Cell Line; D019765:Chondroitin ABC Lyase / Q000494:pharmacology; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000187:drug effects* / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006497:Heparan Sulfate / Q000096:biosynthesis; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012972:Sodium Hydroxide / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism
D020126:Brefeldin A; D019765:Chondroitin ABC Lyase; D011508:Chondroitin Sulfate Proteoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D010724:Phosphoadenosine Phosphosulfate; D011500:Protein Synthesis Inhibitors; D012972:Sodium Hydroxide