Mutant prion proteins axe partially retained in the endoplasmic reticulum

Familial prion diseases are linked to point and insertional mutations in th e prion protein (PrP) gene that are presumed to favor conversion of the cel lular isoform of PrP to the infectious isoform. In this report, we have inv estigated the subcellular localization of PrP molecules carrying pathogenic mutations using immunofluorescence staining, immunogold labeling, and PrP- green fluorescent protein chimeras. To facilitate visualization of the muta nt proteins, we have utilized a novel Sindbis viral replicon engineered to produce high protein levels without cytopathology. We demonstrate that seve ral different pathogenic mutations have a common effect on the trafficking of PrP, impairing delivery of the molecules to the cell surface and causing a portion of them to accumulate in the endoplasmic reticulum. These observ ations suggest that protein quality control in the endoplasmic reticulum. m ay play an important role in prion diseases, as it does in some other inher ited human disorders. Our experiments also show that chimeric PrP molecules with the sequence of green fluorescent protein inserted adjacent to the gl ycolipidation site are post-translationally modified and localized normally , thus documenting the utility of these constructs in cell biological studi es of PrP.