Retrotranslocation of prion proteins from the endoplasmic reticulum by preventing GPI signal transamidation

Abstract

Neurodegeneration in diseases caused by altered metabolism of mammalian prion protein (PrP) can be averted by reducing PrP expression. To identify novel pathways for PrP down-regulation, we analyzed cells that had adapted to the negative selection pressure of stable overexpression of a disease-causing PrP mutant. A mutant cell line was isolated that selectively and quantitatively routes wild-type and various mutant PrPs for ER retrotranslocation and proteasomal degradation. Biochemical analyses of the mutant cells revealed that a defect in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an unprocessed GPI-anchoring signal sequence that directs both ER retention and efficient retrotranslocation of PrP. An unprocessed GPI signal was sufficient to impart ER retention, but not retrotranslocation, to a heterologous protein, revealing an unexpected role for the mature domain in the metabolism of misprocessed GPI-anchored proteins. Our results provide new insights into the quality control pathways for unprocessed GPI-anchored proteins and identify transamidation of the GPI signal sequence as a step in PrP biosynthesis that is absolutely required for its surface expression. As each GPI signal sequence is unique, these results also identify signal recognition by the GPI-transamidase as a potential step for selective small molecule perturbation of PrP expression.

Figure 1.

Altered PrP expression in the A4 mutant cell line. (A) Total cell lysates from N2a cells transiently transfected with either wild type (WT) or the A117V PrP mutant were separated into detergent soluble (S) and insoluble (P) fractions, resolved by SDS-PAGE, and immunoblotted using an antibody (3F4) that specifically detects transfected but not endogenous PrP. The migration of different PrP species are indicated: Mat, mature PrP containing fully modified glycans; Imm, Immature PrP containing core or incompletely modified glycans; −CHO, unglycosylated PrP. Bottom panels, the corresponding indirect immunofluorescence images using the 3F4 antibody. (B) Immunoblot analysis of the parental N2a cells and two derived cell lines (C3 and A4) that stably express WT or PrP(A117V), respectively. The left panel was probed with 3F4 to detect expression of the stably transfected product, and the right panel with PrP-A (a pan-PrP antibody) to detect both endogenous and transfected products. (C) Indirect immunofluorescent localization of PrP in C3 and A4 cells using the 3F4 antibody. Identical detector settings were used to allow comparison of relative expression levels. The lower panels show single cells to illustrate the different PrP localization patterns in C3 versus A4 cells, the latter of which colocalized with RFP-KDEL, an ER marker introduced by transient transfection. (D) A4 and parental N2a cells were transiently transfected with plasmids encoding either WT or A117V PrP and analyzed by fractionation and 3F4 immunoblotting as in A. (E) Total cell lysates from A4 and N2a cells transiently transfected with WT PrP were digested with EndoH (E), with PNGase F (P) or left untreated (−) before immuoblotting with 3F4. The lower panel shows this immunoblot stripped and reprobed with an antibody against TRAPα, an ER resident glycoprotein. (F) A4 and parental N2a cells were transiently transfected with WT PrP and analyzed by indirect immunofluorescence using 3F4. Note the lack of surface expression in A4 cells, in which all of the PrP colocalized with RFP-KDEL (bottom panels).

Figure 2.

Constitutive ERAD of PrP in A4 cells. (A) Pulse-chase analysis of the stably transfected PrP products (immunoprecipitated using 3F4) in either A4 or C3 cells. Pulse labeling with [³⁵S]methionine was for 10 min, followed by chase for the indicated times (in hours). (B and C) A4 and parental N2a cells were transiently transfected with the indicated constructs and analyzed by pulse chase as in A. The samples in B were immunoprecipitated with 3F4, whereas the HA-tagged proteins in C were recovered using anti-HA antibody. (D) Pulse-chase analysis of A4 cells performed in the absence or presence of a proteasome inhibitor (5 μM MG132) or mannosidase I inhibitor (1 μM kifunensine). (E) Steady-state levels of PrP in A4 cells (detected using 3F4) after treatments with MG132 or kifunensine for the indicated times (in hours). (F) Pulse-chase analysis of A4 cells transiently transfected with the indicated mutant PrPs performed in the absence or presence of proteasome inhibitor (5 μM MG132). (G) Indirect immunofluorescence (using 3F4) of A4 cells before and after treatment with 5 μM MG132 for 4 h.

Figure 3.

Normal glycoprotein metabolism in A4 cells. (A) The synthesis and turnover of total glycoproteins (captured using immobilized concanavalin-A) was assessed in A4 and N2a cells using pulse-chase analysis. Pulse labeling with [³⁵S]methionine was for 15 min, followed by chase for the indicated times (in hours). The positions of molecular weight markers (in kDa) are indicated on the left. (B) Synthesis and turnover of VSVG-GFP and ETBR-GFP in A4 and parental N2a cells was followed by pulse-chase analysis as in Figure 2A. Immunoprecipitation was with anti-GFP antibody. (C) Representative fluorescence images of A4 and N2a cells transfected with VSVG-GFP and ETBR-GFP.

Figure 4.

The GPI-anchoring signal sequence is necessary for ERAD of PrP in A4 cells. (A) The turnover of GFP-GPI_FR was analyzed in transiently transfected A4 and N2a cells by pulse-chase labeling and immunoprecipitation (with anti-GFP). Pulse labeling was for 15 min, followed by chase for the indicated times (in hours). (B) Left, fluorescence images of A4 and N2a cells transfected with GFP-GPI_FR; right, enlarged views of A4 and N2a cells cotransfected with GFP-GPI_FR (in green) and RFP-KDEL (in red). Yellow indicates colocalization of the two proteins. (C) The metabolism of PrP lacking the GPI-anchoring signal sequence (PrP-ΔGPI) was determined in A4 and parental N2a cells by pulse-chase analysis followed by immunoprecipitation of cell lysates (L) and culture media (M) with 3F4. Labeling was for 10 min, followed by chase for either 0 or 6 h. The positions of stably expressed PrP(A117V) and transiently transfected PrP-ΔGPI are indicated by the single and double asterisks, respectively. (D) Steady-state levels of PrP-Qa in N2a and A4 cells (detected using 3F4) in the presence (+) or absence (−) of MG132 for 6 h. (E) Unglycosylated PrP from tunicamycin-treated A4 cells was compared in its migration to in vitro–synthesized full-length PrP (FL), PrP lacking the N-terminal signal sequence (ΔSS), or PrP lacking both the N- and C-terminal signals (Δ/Δ). PrP was detected by immunoblotting using the 3F4 antibody.

Figure 5.

A4 cells are defective in GPI signal processing rather than post-ER trafficking. (A) Pulse-chase analysis of transiently transfected WT PrP in N2a cells in the absence (−) or presence (+) of 10 μg/ml brefeldin A (BFA), a drug that prevents ER-to-Golgi trafficking. (B) Total lysates (T) from untransfected A4 and N2a cells were prepared in TX-114 containing buffer and separated into aqueous (A) and hydrophobic (H) phases. These samples were immunoblotted with the PrP-A antibody. Note that recovery of material from the hydrophobic phase is less efficient under our assay conditions. The asterisk denotes a nonspecific soluble protein detected by the PrP-A antibody that serves as a control for complete and comparable recovery of proteins from the aqueous phase of both cell types.

Figure 6.

Defects in GPI anchor biosynthesis in A4 and L-cells routes PrP for ERAD. (A) A4, N2a, and GPI anchor-deficient L-cells were labeled with [³H]mannose for 2 h, extracted with organic solvent to isolate the glycolipid fraction, and analyzed by TLC and autoradiography. The arrowhead points to the fully mature GPI lipid anchor, the major mannose-containing lipid in N2a cells that is lacking in both A4 and L-cells. The darker exposure on the right revealed several presumably immature mannolipid species (asterisks) present in A4 cells that are not found in L-cells. O, origin; F, solvent front. (B) Total cell lysates from untransfected A4, N2a, C3 and L-cells were immunoblotted using the PrP-A antibody to detect steady-state levels of endogenously expressed PrP. (C) Biosynthesis and maturation of endogenous PrP in A4 and L-cells were assessed by pulse-chase analysis followed by immunoprecipitation using the PrP-A antibody. Pulse labeling with [³⁵S]methionine was for 10 min, followed by chase for the indicated times (in hours). (D) Steady-state levels of PrP in L-cells (detected using PrP-A antibody) after treatments with 5 μM MG132 or 1 μM kifunensine for the indicated times (in hours). (E) Steady-state levels of PrP(S232W) in N2a cells (detected using 3F4) after treatments with 5 μM MG132 (M) or 1 μM kifunensine (K) for 4 h. U, untreated cell lysates. Untreated lysates from WT PrP expressing cells are shown for comparison (WT). (F) Pulse-chase analysis of WT and PrP(S232W) in N2a cells performed in the absence (−) or presence (+) of 5 μM MG132.

Figure 7.

The mature domain influences the fate of proteins with unprocessed GPI signals. (A) Pulse-chase analysis in A4 cells of GFP-GPI_FR or its glycosylated counterpart (GFP-GPI-CHO) in the absence (Unt.) or presence of 5 μM MG132 (MG.) or 1 μM kifunensine (Kif.). Pulse labeling was for 10 min and chase for either 0 or 6 h. The percent of protein degraded by 6 h was quantified by phosphorimaging and is indicated below each set of lanes. (B) Pulse-chase analysis of PrP turnover in A4 cells in the absence or presence of 10 μg/ml the glycosylation inhibitor tunicamycin. (C) Pulse-chase analysis of GFP-GPI_PrP (left panels) and GFP-GPI_FR (right panels) in A4 cells. Top panels, PrP immunoprecipitations using the 3F4 antibody; bottom panels, GFP immunoprecipitations. (D) Pulse-chase analysis of transiently transfected WT PrP in A4 and N2a cells in the absence (Unt.) or presence of the reversible reducing agent, dithiothreitol (DTT; 10 mM). +/−DTT indicates samples that were labeled in the presence, but chased in the absence of DTT. The right panel shows the turnover of WT PrP in A4 cells in the absence (Unt.) or presence of ER stress induced by thapsigargin treatment (Tg; 1 μM). (E) Metabolically labeled cell lysates from A4 cells transfected with GFP-GPI_FR were immunoprecipitated under nondenaturing conditions using either the PrP-A antibody (left lane) or an anti-GFP antibody (right lane), to detect coassociating proteins. The arrowhead points to the migration of PrP and GFP. Asterisks mark coassociating proteins that are enriched in either the PrP or GPI-GFP immunoprecipitates. (F) Metabolically labeled A4 cell lysates were immunoprecipitated with PrP-A, anti-GFP, or anti-protein disulfide isomerase (PDI) antibodies under nondenaturing conditions (1st IP) followed by denaturation and reimmunoprecipitation with the PrP-A antibody (2nd IP). The arrowhead shows the presence of PrP in the sample that was sequentially immunoprecipitated with anti-PDI and PrP-A antibodies.

Figure 8.

Working model for the regulation of PrP degradation and trafficking by GPI signal sequence transamidation. (1) Interaction of PrP with PDI (green) during cotranslational translocation into the ER. (2) Recognition of and interaction with the GPI-anchoring signal sequence (red) by the GPI transamidase enzyme complex (blue) leads to the replacement of the GPI signal sequence with a preassembled GPI lipid anchor in cells (e.g., N2a) where GPI anchor biosynthesis is intact (3). This lipid anchored PrP species is now competent for ER exit. PrP species that contain a GPI signal sequence that is inserted into the ER membrane as a transmembrane segment and anchorless PrP generated by transamidation of the signal by a nucleophile such as water are also species that remain competent for trafficking out of the ER. (4) In cells defective in GPI anchor synthesis (e.g., A4 and L-cells) the GPI signal-containing form of PrP may maintain prolonged interactions with chaperones such as PDI. (5) These interactions eventually deliver misprocessed PrP to the ERAD pathway, perhaps through interaction of PDI with the Derlin associated retrotranslocation machinery.