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

The single disulfide loop (Cys(178)-Cys(213)) Of the prion protein (PrP) ma y stabilize the conformation of this protein by bridging the alpha-terminal cr-helices, The substitution mutant Cys(178)Ala fails to form the prion is oform PrPSc when expressed in scrapie-infected neuroblastoma ScN2a cells (M uramoto 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 Delta 2 3-88 version. The resulting mutants (C178A and Delta C178A, respectively) w ere transiently expressed in N2a and CHO cells. Wild-type PrP, wild-type De lta 23-88 and the point mutant E199K served as controls in these experiment s. Compared to the wild-type controls, the C178A mutants were markedly resi stant to proteolysis and they were also vastly insoluble in sarcosyl, Study ing the metabolic fate of the C178A mutants, we found that in contrast to c ontrol PrP molecules, these mutants (i) remained sensitive to the diagnosti c endoglycosidase EndoH, (ii) failed to reach the cell surface and (iii) co ngregated in large juxtanuclear spots. We surmise that these severe traffic king abnormalities may contribute both to the spontaneous aggregation of th e C178A mutants and to their reported inability to form PrPSc. (C) 1999 Fed eration of European Biochemical Societies.