INTRACELLULAR DEGRADATION OF SECRETION DEFECT-TYPE MUTANTS OF ANTITHROMBIN IS INHIBITED BY PROTEASOMAL INHIBITORS

To examine the cellular basis for secretion defect-type antithrombin d eficiency, we expressed two mutants, P --> stop (Pro(429) to stop codo n) and Delta Glu (deletion of Glu(313)), Pulse-chase experiments using stably transfected BHK cells showed that little (< 5%) of P --> stop mutant as well as Delta Glu mutant was secreted and the total amount o f radioactivity was significantly reduced, suggesting an intracellular degradation, The degradation was not inhibited by brefeldin A, indica ting it occurring in a preGolgi apparatus, However, the degradation wa s strongly inhibited by proteasomal inhibitors, such as carbobenzoxy-L -leucyl-L-leucyl-L-leucinal (LLL), carbobenzoxy-L-leucyl-L-leucyl-L-no rvalinal (LLnV) and lactacystin, By endoglycosidase H digestion and im munofluorescence staining, these mutants were shown to localize in the endoplasmic reticulum (ER), These results suggest that the secretion defect-type mutants of antithrombin are degraded by proteasome through the ER-associated quality control mechanism in the cells. (C) 1997 Fe deration of European Biochemical Societies.