Mutant PrP Is Delayed in Its Exit from the Endoplasmic Reticulum, but Neither Wild-type nor Mutant PrP Undergoes Retrotranslocation Prior to Proteasomal Degradation

Abstract

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.

Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells

“…This species, also detected in PC12 and N2a cells, appeared to be similar to that described in earlier studies (Ma & Lindquist, 2001;Cohen and Taraboulos, 2003;Wang et al, 2005). However, we also observed clear accumulation of PrP 26K in primary cultured cortical neurons derived from wild-type mice and treated with ALLN or MG132, which is in contrast to earlier findings in cerebellar neurons (Drisaldi et al, 2003). The reason for this discrepancy is unclear, although it is conceivable that different neuron subpopulations may be variably susceptible to these compounds.…”

“…It has been further proposed that enhanced accumulation of cytosolic PrP might be detrimental to the neurons, and that perturbation of PrP metabolism through the proteasomal pathway could play a pathogenic role in prion diseases (Ma & Lindquist, 1999;Yedidia et al, 2001;Grenier et al, 2006;Wang et al, 2006). For other authors, however, these observations have arisen essentially from experimental settings (Drisaldi et al, 2003;Fioriti et al, 2005). In addition, PrP was found in the cytosol in subpopulations of neurons in several areas of normal brain (Mironov et al, 2003), and a neurotoxic activity of cytosolic PrP C could not be demonstrated in various neuronal cell systems (Roucou et al, 2003;Fioriti et al, 2005).…”

“…Importantly, we found no evidence for the presence of an uncleaved signal peptide on the PrP 26K molecules produced in ALLN-treated CAD and N2A cells. The absence of a detectable signal peptide on insoluble PrP C argues that it was essentially mature protein, rather than a neo-translated protein, that failed to achieve ER translocation as proposed for cells overexpressing tagged PrP driven by a heterologous promoter (Drisaldi et al, 2003;Fioriti et al, 2005).…”

Genetic Mapping of Activity Determinants within Cellular Prion Proteins

“…For some experiments Tg(SHaPrP)7 mice maintained in a B6 congenic background (35) (a gift from G. Carlson) or non-Tg littermates were also used as a source of donor cells. Primary cultures of cerebellar granule cells were prepared from P6 -7-day animals as described previously (7,36). After 4 days in vitro, granule cells were transfected in minimum essential medium with 2 g of pBud-GFP vectors using Lipofectamine 2000 (Invitrogen) for 30 min before replacing the media with K25ϩS.…”

Protein Quality Control in Health and Disease

“…1I). Molecules that fail to enter the ER are degraded by the proteasome (II) (Drisaldi et al 2003). Upon entry into the ER, the signal peptide is cleaved by a signal peptidase (III), oligosaccharides are attached to asparagine residues of the molecule, and a glycosylphosphatidylinositol (GPI) anchor is added to the protein's C-terminus (IV) (Stahl et al 1990).…”