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Mutant prion proteins are partially retained in the endoplasmic reticulum
L Ivanova1, S Barmada, T Kummer
1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Abstract:
Familial prion diseases are linked to point and insertional mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP to the infectious isoform. In this report, we have investigated 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 mutant proteins, we have utilized a novel Sindbis viral replicon engineered to produce high protein levels without cytopathology. We demonstrate that several 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 observations suggest that protein quality control in the endoplasmic reticulum may play an important role in prion diseases, as it does in some other inherited human disorders. Our experiments also show that chimeric PrP molecules with the sequence of green fluorescent protein inserted adjacent to the glycolipidation site are post-translationally modified and localized normally, thus documenting the utility of these constructs in cell biological studies of PrP.
Insights
Pathogenic mutations in the prion protein (PrP) gene impair its trafficking, causing accumulation in the endoplasmic reticulum and suggesting a role for protein quality control in familial prion diseases.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Familial prion diseases are associated with mutations in the prion protein (PrP) gene.
- These mutations are thought to promote the conversion of cellular PrP to infectious forms.
- Understanding the subcellular localization of mutant PrP is crucial for elucidating disease mechanisms.
Purpose of the Study:
- To investigate the subcellular localization of prion protein (PrP) molecules with pathogenic mutations.
- To determine the common effects of different pathogenic mutations on PrP trafficking.
- To explore the role of endoplasmic reticulum protein quality control in prion diseases.
Main Methods:
- Immunofluorescence staining and immunogold labeling were used to visualize PrP.
- PrP-green fluorescent protein (GFP) chimeras were employed to track protein localization.
- A novel Sindbis viral replicon system was utilized for high-level protein expression without cytopathology.
Main Results:
- Several pathogenic PrP mutations were found to impair the trafficking of PrP molecules.
- Mutant PrP showed accumulation in the endoplasmic reticulum, indicating defective transport.
- PrP-GFP chimeras near the glycolipidation site were correctly processed and localized, validating their use.
Conclusions:
- Defective trafficking and endoplasmic reticulum accumulation are common consequences of pathogenic PrP mutations.
- Endoplasmic reticulum protein quality control mechanisms may be critical in the pathogenesis of prion diseases.
- PrP-GFP chimeras are reliable tools for studying PrP cell biology and the impact of mutations.