Mutant Prion Proteins Are Partially Retained in the Endoplasmic Reticulum

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 PrPgreen 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.

Cytosolic Prion Protein (PrP) Is Not Toxic in N2a Cells and Primary Neurons Expressing Pathogenic PrP Mutations

“…Pulse-chase labeling experiments indicated that mutant PrP molecules misfold very soon after synthesis in the endoplasmic reticulum (ER) (6), raising the possibility that they are recognized as abnormal by the ER quality control machinery, and diverted to ER-associated degradation (ERAD). Consistent with this hypothesis, it was observed that several mutant PrPs were present at low levels on the cell surface, and localized in intracellular compartments, including the ER and cytoplasm (7)(8)(9)(10). It was also found that the proteasome inhibitor ALLN (Ac-Leu-Leu-NorLeu-al) affected the metabolism and cellular localization of PrP molecules carrying the amber mutation Y145stop, and the Q217R substitution linked to GSS (11,12).…”

“…In cultured, non-neuronal cells mouse PrP molecules carrying the D177N and nine-octapeptide mutations adopt an abnormal conformation soon after synthesis in the ER (6), and at the steady state they appear to accumulate in the ER and Golgi because their transit out of these organelles is delayed (9,16). Here we provide evidence that the same holds true for mutant PrPs synthesized in primary neurons.…”

“…Neurons-We have previously reported that PG14 and D177N PrPs expressed in transfected cells are delayed in their export from the ER, and that at the steady state they are present on the cell surface at reduced levels compared with wild-type PrP, and accumulate in intra- cellular compartments, including the ER and Golgi (9,16). To investigate the distribution of these mutants in primary neurons, we performed immunofluorescence confocal analysis of PrP in cerebellar granule neurons (CGN) from Tg mice.…”

Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation

“…But studies with a series of mutants associated with transmissible spongiform encephalopathies indicated that most mutants that reside outside the hydrophobic region do not form detectable transmembrane forms (9). Instead, they are partially retained in the ER, which suggests misfolding and retention by the ER quality-control system (10). Recent understanding of protein folding and quality-control mechanisms has revealed that many misfolded secretory proteins are retained in the ER and subject to retrograde transport to the cytoplasm and degradation by the proteasome (11)(12)(13).…”

Prion Neurotoxicity: Insights from Prion Protein Mutants

“…These studies have revealed that some, but not all mutants, display PrP Sc -like biochemical properties including aggregation propensity as well as resistance to proteases and a GPI-specific phospholipase (Lehmann and Harris, 1996;Priola and Chesebro, 1998). Correlating with these abnormal biochemical properties, some mutants display altered subcellular localization, including partial retention in the ER (Ivanova et al, 2001), or retrotranslocation into the cytoplasm with subsequent degradation by the proteasome (Lorenz et al, 2002). Interestingly, some disease-associated PrP mutants are identical to wild-type PrP in terms of their biochemical properties and cellular distribution (Harris, 2003), raising the possibility that these molecules are pathogenic, not because they misfold and aggregate, but because the mutation subtly alters a physiological activity normally associated with PrP C .…”