Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop

The single disulfide loop (Cys₁₇₈-Cys₂₁₃) of the prion protein (PrP) may stabilize the conformation of this protein by bridging the C-terminal α-helices. The substitution mutant Cys₁₇₈Ala fails to form the prion isoform PrP(Sc) when expressed in scrapie-infected neuroblastoma ScN2a cells (Muramoto et al., Proc. Natl. Acad. Sci. USA 93, 15457-15462). To investigate the reasons for this failure, we introduced the C178A substitution in the full length mouse PrP gene as well as in its N-terminally truncated Δ23-88 version. The resulting mutants (C178A and ΔC178A, respectively) were transiently expressed in N2a and CHO cells. Wild-type PrP, wild-type Δ23-88 and the point mutant E199K served as controls in these experiments. Compared to the wild-type controls, the C178A mutants were markedly resistant to proteolysis and they were also vastly insoluble in sarcosyl. Studying the metabolic fate of the C178A mutants, we found that in contrast to control PrP molecules, these mutants (i) remained sensitive to the diagnostic endoglycosidase EndoH, (ii) failed to reach the cell surface and (iii) congregated in large juxtanuclear spots. We surmise that these severe trafficking abnormalities may contribute both to the spontaneous aggregation of the C178A mutants and to their reported inability to form PrP(Sc). Copyright (C) 1999 Federation of European Biochemical Societies.