An amber mutation at codon 145 (Y145stop) of the prion protein gene results in a variant of an inherited human prion disease named Gerstmann- Straussler-Scheinker syndrome. The characteristic features of this disorder include amyloid deposits of prion protein in cerebral parenchyma and vessels. We have studied the biosynthesis and processing of the prion protein containing the Y145stop mutation (PrP145) in transfected human neuroblastoma cells in an attempt to clarify the effect of the mutation on the metabolism of PrP145 and to gain insight into the underlying pathogenetic mechanism. Our results demonstrate that 1) a significant proportion of PrP145 is not processed post-translationally and retains the N-terminal signal peptide, 2) most PrP145 is degraded very rapidly by the proteasome-mediated pathway, 3) blockage of proteasomal degradation results in intracellular accumulation of PrP145, 4) most of the accumulated PrP145 is detergent-insoluble, and both the detergent-soluble and - insoluble fractions are resistant to mild proteinase K (PK) treatment, suggesting that PK resistance is not simply because of aggregation. The present study demonstrates for the first time that a mutant prion protein is degraded through the proteasomal pathway and acquires PK-resistance if degradation is impaired.