IDENTIFICATION OF INTERMEDIATE STEPS IN THE CONVERSION OF A MUTANT PRION PROTEIN TO A SCRAPIE-LIKE FORM IN CULTURED-CELLS

The central causative event in infectious, familial, and sporadic form s of prion disease is thought to be a conformational change that conve rts the cellular isoform of the prion protein (PrPC) into the scrapie isoform (PrPSc) that is the primary constituent of infectious prion pa rticles, To provide a model system for analyzing the mechanistic detai ls of this critical transformation, we have previously prepared cultur ed Chinese hamster ovary cells that stably express mouse PrP molecules carrying mutations homologous to those seen in familial prion disease s of humans, In the present work, we have analyzed the kinetics with w hich a PrP molecule containing an insertional mutation associated with Creutzfeldt-Jakob disease acquires several biochemical properties cha racteristic of PrPSc. Within 10 min of pulse labeling, the mutant prot ein undergoes a molecular alteration that is detectable by a change in Triton X-114 phase partitioning and phenyl-Sepharose binding. After 3 0 min of labeling, a detergent-insoluble and protease-sensitive form o f the protein appears, After a chase period of several hours, the prot ein becomes protease-resistant. Incubation of cells at 18 degrees C or treatment with brefeldin A inhibits acquisition of detergent insolubi lity and protease resistance but does not affect Triton X-114 partitio ning and phenyl-Sepharose binding, Our results support a model in whic h conversion of mutant PrPs to a PrPSc-like state proceeds in a stepwi se fashion via a series of identifiable biochemical intermediates, wit h the earliest step occurring during or very soon after synthesis of t he polypeptide in the endoplasmic reticulum.