Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation - PubMed (original) (raw)

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

J Ma et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The cytoplasm seems to provide an environment that favors conversion of the prion protein (PrP) to a form with the physical characteristics of the PrP(Sc) conformation, which is associated with transmissible spongiform encephalopathies. However, it is not clear whether PrP would ever exist in the cytoplasm under normal circumstances. We report that PrP accumulates in the cytoplasm when proteasome activity is compromised. The accumulated PrP seems to have been subjected to the normal proteolytic cleavage events associated with N- and C-terminal processing in the endoplasmic reticulum, suggesting that it arrives in the cytoplasm through retrograde transport. In the cytoplasm, PrP forms aggregates, often in association with Hsc70. With prolonged incubation, these aggregates accumulate in an "aggresome"-like state, surrounding the centrosome. A mutant (D177N), which is associated with a heritable and transmissible form of the spongiform encephalopathies, is less efficiently trafficked to the surface than wild-type PrP and accumulates in the cytoplasm even without proteasome inhibition. These results demonstrate that PrP can accumulate in the cytoplasm and is likely to enter this compartment through normal protein quality-control pathways. Its potential to accumulate in the cytoplasm has implications for pathogenesis.

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Figures

Figure 1

Figure 1

Effect of proteasome inhibition on endogenous PrP in NT-2 cells. Cells were treated without (control) or with MG132 at indicated concentrations for 12 hr. (A) Merged images of cells costained with anti-PrP antibody (red) and Lucifer yellow CH (green, lysosome staining) (Upper) or with anti-PrP (red) and anti-Hsc70 antibodies (green) (Lower). (B, Upper) MG132-treated cells were stained with anti-PrP antibody (red) and Lucifer yellow CH (green). The regions outlined by dashed boxes were merged and magnified (Right). (Lower) MG132-treated cells were stained with anti-PrP antibody (red) and anti-Hsc70 antibody (green).

Figure 2

Figure 2

Physical state of PrP in NT-2 cells with or without proteasome inhibition. (A) Detergent cell lysates were sedimented, and PrP in the supernatant and pellet fractions was detected by immunoblot analysis with anti-PrP 3F4 monoclonal antibody. (B) Comigration of PrP from the pellet fraction with the recombinant mature fragment of PrP-(23–230), which lacks both N- and C-terminal signal sequences.

Figure 3

Figure 3

Effect of proteasome inhibitor on PrP localization in COS cells. Cells expressing wild-type PrP were treated without (A) or with 5 μM epoxomicin for various lengths of time as indicated in_B–D_. (E) Cells without proteasome inhibition but overexpressing PrP because of high transfection levels. In all panels, PrP was stained with anti-PrP antibody (green). Giantin, hsc70, or γ-tubulin (red) staining with specific antibodies was indicated; nuclei were stained with DAPI (blue). All of the cells were fixed by methanol except for visualization of surface staining in cells fixed by paraformaldehyde. Large arrows in E, cells with cytoplasmic PrP colocalizing with Hsc70; small arrows in_E_, cells with PrP in the Golgi complex. (E, Right) Higher magnification of colocalization of PrP aggregates and hsc70.

Figure 4

Figure 4

The physical state of PrP in transfected cells. (A) COS cells transfected with wild-type PrP were incubated with or without 1 μM epoxomicin for 16 hr. Detergent lysates were fractionated by centrifugation, and PrP in the supernatant (Sup) or pellet fractions was detected by immunoblot analysis using 3F4 antibody. (B) PrP in the pellet fraction was detected by immunoblot analysis with antibodies specific for the N-terminal (R24) or C-terminal (R20) regions of mature PrP as indicated. EndoH, PrP in the Endo H-digested pellet fraction was detected by 3F4 antibody.

Figure 5

Figure 5

Effect of the D177N mutation on PrP localization. COS cells were transfected with plasmids encoding either wild-type PrP or the D177N mutant. (A) Surface staining of paraformaldehyde-fixed cells expressing wild-type PrP (WT) or PrPD177N (D177N) by using 3F4 antibody. (B) Intracellular staining of PrPD177N. Cells were costained with 3F4 antibody and anti-BiP antibody (Top), Lucifer yellow CH (endocytic compartments) (Middle), or anti-Hsc70 antibody (Bottom). (C) Cells expressing PrPD177N were treated with 5 μM epoxomicin for 16 hr and costained with 3F4 and anti-Hsc70 antibodies.

Figure 6

Figure 6

Physical state of PrPD177. COS cells were transfected as in Fig. 4. The activity of a cotransfected β-galactosidase construct (data not shown) demonstrated equivalent transfection efficiency. PrP was detected by 3F4 antibody. C, vector control; WT, PrP; M, PrPD177. (A, Left) Total PrP in lysates. (Right) PrP in Endo H-digested lysates. (B) PrP in supernatant and pellet fractions of cell lysates. The same blots were also probed with calnexin antibody.

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