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• W2017014906 abstract "Aberrant folding of the mammalian prion protein (PrP) is linked to prion diseases in humans and animals. We show that during post-translational targeting of PrP to the endoplasmic reticulum (ER) the putative transmembrane domain induces misfolding of PrP in the cytosol and interferes with its import into the ER. Unglycosylated and misfolded PrP with an uncleaved N-terminal signal sequence associates with ER membranes, and, moreover, decreases cell viability. PrP expressed in the cytosol, lacking the N-terminal ER targeting sequence, also adopts a misfolded conformation; however, this has no adverse effect on cell growth. PrP processing, productive ER import, and cellular viability can be restored either by deleting the putative transmembrane domain or by using a N-terminal signal sequence specific for co-translational ER import. Our study reveals that the putative transmembrane domain features in the formation of misfolded PrP conformers and indicates that post-translational targeting of PrP to the ER can decrease cell viability. Aberrant folding of the mammalian prion protein (PrP) is linked to prion diseases in humans and animals. We show that during post-translational targeting of PrP to the endoplasmic reticulum (ER) the putative transmembrane domain induces misfolding of PrP in the cytosol and interferes with its import into the ER. Unglycosylated and misfolded PrP with an uncleaved N-terminal signal sequence associates with ER membranes, and, moreover, decreases cell viability. PrP expressed in the cytosol, lacking the N-terminal ER targeting sequence, also adopts a misfolded conformation; however, this has no adverse effect on cell growth. PrP processing, productive ER import, and cellular viability can be restored either by deleting the putative transmembrane domain or by using a N-terminal signal sequence specific for co-translational ER import. Our study reveals that the putative transmembrane domain features in the formation of misfolded PrP conformers and indicates that post-translational targeting of PrP to the ER can decrease cell viability. Prion diseases in humans and animals are characterized by the accumulation of PrPSc, a partially protease-resistant isoform of the cellular prion protein PrPC. PrPSc is generated through a conformational transformation of PrPC and represents the major component of infectious prions (reviewed in Refs. 1Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar, 2Aguzzi A. Montrasio F. Kaeser P.S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 118-126Crossref PubMed Scopus (132) Google Scholar, 3Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1110) Google Scholar, 4Weissmann C. Fischer M. Raeber A. Büeler H. Sailer A. Shmerling D. Rülicke T. Brandner S. Aguzzi A. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 511-522Crossref PubMed Google Scholar). PrP 1The abbreviations used are: PrP, prion protein; PK, proteinase K; TM, transmembrane; ER, endoplasmic reticulum; Endo H, endo-β-N-acetylglucosaminidase H; GPI, glycosylphosphatidylinositol; aa, amino acid(s); SRP, signal recognition particle; SRPR, signal recognition particle receptor; TE, Tris-EDTA. is post-translationally modified by the attachment of two N-linked complex carbohydrate moieties (Asn180 and Asn196) (5Bolton D.C. Meyer R.K. Prusiner S.B. J. Virol. 1985; 53: 596-606Crossref PubMed Google Scholar, 6Endo T. Groth D. Prusiner S.B. Kobata A. Biochemistry. 1989; 28: 8380-8388Crossref PubMed Scopus (254) Google Scholar, 7Haraguchi T. Fisher S. Olofsson S. Endo T. Groth D. Tarentino A. Borchelt D.R. Teplow D. Hood L. Burlingame A. Lycke E. Kobata A. Prusiner S.B. Arch. Biochem. Biophys. 1989; 274: 1-13Crossref PubMed Scopus (186) Google Scholar) and a glycosylphosphatidylinositol (GPI) anchor at serine 231 (8Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (908) Google Scholar) as well as by the formation of a disulfide bond between Cys178 and Cys213. Studies with recombinant PrP purified from bacteria revealed that the formation of the disulfide bond is essential for the native folding of PrP (9Maiti N.R. Surewicz W.K. J. Biol. Chem. 2001; 276: 2427-2431Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The co- and post-translational modifications of PrPC are initiated with the cleavage of the N-terminal signal peptide (aa 1–22) and the transfer of core glycans, whereas the nascent chain is still associated with the translocon. Shortly after the protein is fully translocated, the GPI anchor is attached to the acceptor amino acid close to the C terminus. The final maturation of PrPC includes the processing of the core glycans into complex-type glycans by a series of enzymatic reactions in the endoplasmic reticulum (ER) and Golgi compartment. Post-translational modifications, like N-linked glycosylation and GPI anchor attachment, are often used as diagnostic markers to monitor efficient import into the ER. In the case of PrP, however, we and others have shown that PrP devoid of a GPI anchor remains mainly unglycosylated but is imported efficiently into the ER and transported through the secretory pathway (10Winklhofer K.F. Heske J. Heller U. Reintjes A. Muranji W. Moarefi I. Tatzelt J. J. Biol. Chem. 2003; 278: 14961-14970Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11Walmsley A.R. Zeng F.N. Hooper N.M. EMBO J. 2001; 20: 703-712Crossref PubMed Scopus (67) Google Scholar, 12Rogers M. Yehiely F. Scott M. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3182-3186Crossref PubMed Scopus (158) Google Scholar, 13Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury Jr., P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (791) Google Scholar, 14Blochberger T.C. Cooper C. Peretz D. Tatzelt J. Griffith O.H. Baldwin M.A. Prusiner S.B. Protein Eng. 1997; 10: 1465-1473Crossref PubMed Scopus (38) Google Scholar). It has been found that the only specific marker for ER import of PrP is a cleaved N-terminal signal sequence (10Winklhofer K.F. Heske J. Heller U. Reintjes A. Muranji W. Moarefi I. Tatzelt J. J. Biol. Chem. 2003; 278: 14961-14970Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). 2U. Heller, K. F. Winklhofer, J. Heske, A. Reintjes, and J. Tatzelt, unpublished results. Misfolding of PrPC in the cytosol or in the ER can induce neurodegeneration in the absence of PrPSc. Neurotoxic properties of cytosolic PrP aggregates were observed after proteasomal inhibition in cultured cells or after the forced expression of cytosolic PrP in transgenic mice (15Ma J. Wollmann R. Lindquist S. Science. 2002; 298: 1781-1785Crossref PubMed Scopus (430) Google Scholar). Other studies revealed that a minor fraction of PrP can be synthesized as an integral membrane protein with two different topologies, termed either NtmPrP, with the N terminus facing the ER lumen, or CtmPrP, with the C terminus facing the ER lumen. Amino acids 112–135 of PrP were identified as a putative transmembrane domain (TM) (16Yost C.S. Lopez C.D. Prusiner S.B. Myers R.M. Lingappa V.R. Nature. 1990; 343: 669-672Crossref PubMed Scopus (109) Google Scholar), and mutations in the TM domain were found to alter the relative amount of CtmPrP and NtmPrP (17Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar). Remarkably, the increased synthesis of CtmPrP has been shown to coincide with progressive neurodegeneration both in Gerstmann-Sträussler-Scheinker syndrome patients with an A117V mutation and in transgenic mice carrying a triple mutation within the putative TM domain (AV3) (17Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar). Other studies on the putative TM domain indicated that the hydrophobic stretch of amino acids 106 to 126 has the propensity to form fibrils (18Tagliavini F. Prelli F. Verga L. Giaccone G. Sarma R. Gorevic P. Ghetti B. Passerini F. Ghibaudi E. Forloni G. Salmona M. Bugiani O. Frangione B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9678-9682Crossref PubMed Scopus (235) Google Scholar) and can induce cell death in cultured cells (19Deli M.A. Sakaguchi S. Nakaoke R. Abraham C.S. Takahata H. Kopacek J. Shigematsu K. Katamine S. Niwa M. Neuroreport. 2000; 11: 3931-3936Crossref PubMed Scopus (37) Google Scholar, 20Brown D.R. Schmidt B. Kretzschmar H.A. Nature. 1996; 380: 345-347Crossref PubMed Scopus (499) Google Scholar, 21Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, 22Haïk S. Peyrin J.M. Lins L. Rosseneu M.Y. Brasseur R. Langeveld J.P. Tagliavini F. Deslys J.P. Lasmézas C. Dormont D. Neurobiol. Dis. 2000; 7: 644-656Crossref PubMed Scopus (42) Google Scholar). In mammalian cells, secretory proteins are usually translocated into the ER via the co-translational pathway, which requires the binding of the signal recognition particle (SRP) to the nascent protein. The SRP directs the targeting of the whole nascent chain-ribosome complex to its receptor (SRPR) on the ER membrane, resulting in the transfer of the growing protein to the translocation pore and a direct release into the ER lumen as soon as the synthesis has finished. An alternative post-translational import pathway has been described in mammalian cells for proteins of less than 75 amino acids in length; in this case the completely synthesized protein is targeted to the ER independently of the SRP/SRPR system (23Müller G. Zimmermann R. EMBO J. 1987; 6: 2099-2107Crossref PubMed Scopus (71) Google Scholar, 24Schlenstedt G. Zimmermann R. EMBO J. 1987; 6: 699-703Crossref PubMed Scopus (63) Google Scholar). In yeast cells, however, a variety of secretory proteins can be imported post-translationally. Whether a co- or post-translational translocation pathway is used in yeast cells seems to be determined mainly by the N-terminal signal sequence. Signal sequences with a hydrophobicity index (Kyte-Doolittle) higher than 2 are strictly dependent on the SRP/SRPR pathway (25Ng D.T. Brown J.D. Walter P. J. Cell Biol. 1996; 134: 269-278Crossref PubMed Scopus (375) Google Scholar) (reviewed in Refs. 26Fewell S.W. Travers K.J. Weissman J.S. Brodsky J.L. Annu. Rev. Genet. 2001; 35: 149-191Crossref PubMed Scopus (264) Google Scholar, 27Keenan R.J. Freymann D.M. Stroud R.M. Walter P. Annu. Rev. Biochem. 2001; 70: 755-775Crossref PubMed Scopus (480) Google Scholar, 28Pilon M. Schekman R. Cell. 1999; 97: 679-682Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29Walter P. Lingappa V.R. Annu. Rev. Cell Biol. 1986; 2: 499-516Crossref PubMed Scopus (356) Google Scholar, 30Zimmermann R. Biol. Chem. Hoppe-Seyler. 1998; 379: 275-282PubMed Google Scholar). In this study we analyzed the biogenesis of mouse PrP and showed that the putative transmembrane domain induces the misfolding of PrP during post-translational targeting to the ER. As a consequence, unprocessed and misfolded PrP associates with ER membranes and interferes with cell viability. Strains, Cells, Antibodies, and Reagents—Wild-type yeast (MATa, ura3, his4) was grown in rich medium containing 2% dextrose (YPD). Transformed cells were grown in synthetic complete medium (SCD, 2% dextrose, 0.7% yeast nitrogen base) supplemented with strain-specific nutrients at 30 °C. Solid media contained 2.3% agar (Difco). Mouse neuroblastoma (N2a) cells (31Klebe R.J. Ruddle F.H. J. Cell Biol. 1969; 43: 69aGoogle Scholar) were cultured as described previously (32Tatzelt J. Prusiner S.B. Welch W.J. EMBO J. 1996; 15: 6363-6373Crossref PubMed Scopus (270) Google Scholar). PrP was detected by the monoclonal anti-PrP antibody 3F4 (33Serban D. Taraboulos A. DeArmond S.J. Prusiner S.B. Neurology. 1990; 40: 110-117Crossref PubMed Google Scholar) or the polyclonal anti-PrP antiserum A7 (10Winklhofer K.F. Heske J. Heller U. Reintjes A. Muranji W. Moarefi I. Tatzelt J. J. Biol. Chem. 2003; 278: 14961-14970Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The anti-Gas1p antibody was kindly provided by R. Barz, and the anti-Gim2p antibody was described earlier (34Geissler S. Siegers K. Schiebel E. EMBO J. 1998; 17: 952-966Crossref PubMed Scopus (236) Google Scholar). Vacuolar ATPase was detected with the monoclonal anti-V-ATPase antibody 10D7-A7-B2 (Molecular Probes). Renografin 76 was prepared by adjusting TE, pH 7.6, to 66% meglumine diatrizoate and 10% sodium diatrizoate. Chemicals were purchased from Sigma and United States Biochemical. Enzymes were purchased from New England Biolabs and Promega. DNA Manipulations and Transformation—PrP constructs were cloned into pYES2 or pcDNA3.1 (Invitrogen) for expression in yeast and mammalian cells, respectively, using standard procedures. In yeast, expression was driven by the GAS1 promoter (bases -413 to -1). GAS1 sequences were amplified by PCR from pBQ1R14 (R. Barz). PrP sequences were amplified from pcDNA3.1–3F4 (35Gilch S. Winklhofer K.F. Nunziante M. Lucassen R. Spielhaupter C. Muranyi W. Groschup M.H. Riesner D. Tatzelt J. Schätzl H.M. EMBO J. 2001; 20: 3957-3966Crossref PubMed Scopus (156) Google Scholar), which contains the mouse PRNP modified to express PrPL108M/V111M for immunostaining with the monoclonal antibody 3F4. The construct pPrPΔGPI contains aa 1–231. For targeting into the yeast ER, aa 1–27 of PrP containing the endogenous mouse signal were replaced by the ER signal sequences of Gas1p (gPrP constructs, aa 1–26) or Kre5p (kPrP constructs, aa 1–21). In gPrP and kPrP, aa 228–254 containing the mouse GPI signal sequence were replaced by the GAS1 GPI signal sequence (aa 526–559). In gPrPΔGPI and all other PrP constructs destined for expression in yeast, the GPI attachment signal was removed (aa 232–254). In gPrPΔ28–140 and gPrPΔ28–156 the respective PrP sequences were deleted. The deletion in gPrPΔTM spans aa 105–133. Cyto-PrP comprises aa 23–231. gPrP-AV3 was constructed by substituting GTC for GCA-(433–435) and GCT-(439–441) and GTT for GCT-(448–450) in the gPrPΔGPI construct in order to replace the alanines at positions 112, 114, and 117 by valines. In gPrP-G122P, the bases GGG-(463–465) were replaced by CCG to code for proline instead of glycine. Yeast cells were transformed using the LiCl method (36Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Yeast. 1999; 15: 963-972Crossref PubMed Scopus (815) Google Scholar, 37Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1773) Google Scholar). N2a cells were transfected using the LipofectAMINE Plus reagent (Invitrogen) as described previously (38Winklhofer K.F. Reintjes A. Hoener M.C. Voellmy R. Tatzelt J. J. Biol. Chem. 2001; 276: 45160-45167Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Protein Preparation of Yeast Extracts and Endo H Treatment—Yeast cells were grown to midlog phase. 50 OD units were poisoned with 10 mm NaF, and proteins were prepared as described previously (39Schandel K.A. Jenness D.D. Mol. Cell. Biol. 1994; 14: 7245-7255Crossref PubMed Google Scholar, 40Wang Q. Chang A. EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar) with the following modifications. Cells were washed in lysis buffer (phosphate-buffered saline containing 0.5% yeast protease inhibitor mixture; Sigma), resuspended in lysis buffer, and broken using glass beads in the Mini-BeadBeater-8 (Biospec Products). A postnuclear supernatant (cell lysate) was prepared by centrifugation at 1000 × g for 5 min at 4 °C. To assess the glycosylation status of proteins, the lysate was digested with endoglycosidase H (Endo H, NEB) following the manufacturer's instructions. Endo H Digestion and Secretion Analysis in N2a Cells—For Endo H digestion, cell lysates were prepared in 0.5% SDS, boiled for 10 min, and digested with Endo H (NEB) for 1 h at 37 °C (10Winklhofer K.F. Heske J. Heller U. Reintjes A. Muranji W. Moarefi I. Tatzelt J. J. Biol. Chem. 2003; 278: 14961-14970Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). After boiling in Laemmli sample buffer, the lysates were examined by immunoblotting. To analyze the secretion of PrP, cells were cultivated in fresh medium for 3 h at 37 °C. PrP present in the supernatant cell culture medium was precipitated by trichloroacetic acid and analyzed by Western blotting. Western Blotting—Following SDS-PAGE, proteins were transferred onto a nitrocellulose membrane and analyzed as described previously (32Tatzelt J. Prusiner S.B. Welch W.J. EMBO J. 1996; 15: 6363-6373Crossref PubMed Scopus (270) Google Scholar). Colony Assay—Yeast cells were grown to stationary phase. A serial dilution was prepared (5-fold in sterile H2O) and spotted onto selective or rich media plates. Growth was monitored after 2 days of incubation at 30 °C. Membrane Fractionation—Localization of recombinant proteins was analyzed by Renografin density gradient centrifugation (39Schandel K.A. Jenness D.D. Mol. Cell. Biol. 1994; 14: 7245-7255Crossref PubMed Google Scholar, 40Wang Q. Chang A. EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar). Cells were washed three times in sorbitol buffer (0.8 m sorbitol, 50 mm Tris, 10 mm NaF, pH 7.6) and in TE (50 mm Tris, 1 mm EDTA, pH 7.6). Lysates were prepared in TE as described above, and 0.5 ml were mixed with 0.5 ml of 76% Renografin, successively overlaid with 1 ml at 34, 30, 26, and 22% Renografin and centrifuged at 150,000 × g for 20 h at 4 °C. 14 fractions were removed from the top of the gradient. Membranes were diluted 10-fold with TE, pelleted at 100,000 × g for 1 h at 4 °C,and resuspended in Laemmli sample buffer. Cell-free Translation and PK Protection Assay—For in vitro translation the TnT Quick transcription/translation kit (Promega) was used according to the manufacturer's instructions. Briefly, 20 μl of TnT, 1 μg of plasmid-DNA, and 1 μl of [35S]methionine (15 mCi/ml, Amersham Biosciences) were adjusted to 25 μl and incubated for 45 min at 30 °C. For co-translational import 2 μl of canine microsomal membranes (Promega) were included in the reaction. All translation reactions were stopped by the addition of 100 μm emetine (Sigma). For post-translational import the membranes were added after stopping the translation. All samples were incubated for a further 45 min at 30 °C and were then divided into two aliquots. For the PK protection assay, proteinase K (0.5 mg/ml, Roche Applied Science) was added to one aliquot and incubated for 1 h on ice. The protease was then inactivated by the addition of 5 mm phenylmethylsulfonyl fluoride (Sigma) for 5 min on ice. Samples were boiled in Laemmli sample buffer and separated by SDS-PAGE. Gels were fixed for 30 min, soaked in Amplify (Amersham Biosciences) for another 30 min, and dried. Signals were visualized using a phosphorimaging device (FLA-2000, Fuji; AIDA 3.26 software, Raytest, Staubenhardt, Germany). PrP Targeted to the ER Is Unprocessed and Interferes with Yeast Growth—Our aim was to use the yeast model to specifically analyze the import of PrP into the ER. In yeast the hydrophobicity index of the N-terminal signal sequence determines whether a co- or post-translational translocation pathway is used (25Ng D.T. Brown J.D. Walter P. J. Cell Biol. 1996; 134: 269-278Crossref PubMed Scopus (375) Google Scholar). Therefore, we expressed PrP with its own signal peptide (pPrP) and also with the ER-targeting peptide of the endogenous GPI-anchored protein Gas1p (gPrP) (41Nuoffer C. Jeno P. Conzelmann A. Riezman H. Mol. Cell. Biol. 1991; 11: 27-37Crossref PubMed Scopus (177) Google Scholar, 42Conzelmann A. Riezman H. Desponds C. Bron C. EMBO J. 1988; 7: 2233-2240Crossref PubMed Scopus (114) Google Scholar). Both signal sequences have a hydrophobicity index (Kyte-Doolittle) lower than 2, which suggests a post-translational targeting of gPrP and pPrP to the ER (25Ng D.T. Brown J.D. Walter P. J. Cell Biol. 1996; 134: 269-278Crossref PubMed Scopus (375) Google Scholar). gPrP contains the C-terminal Gas1p GPI anchor signal sequence, and further PrP constructs were generated lacking either the GPI anchor attachment signal (gPrPΔGPI, pPrPΔGPI) or lacking both the ER targeting and the GPI anchor attachment signals (cyto-PrP) (Fig. 1A). It should be noted that PrPΔGPI expressed in mammalian cells is completely imported into the ER (10Winklhofer K.F. Heske J. Heller U. Reintjes A. Muranji W. Moarefi I. Tatzelt J. J. Biol. Chem. 2003; 278: 14961-14970Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11Walmsley A.R. Zeng F.N. Hooper N.M. EMBO J. 2001; 20: 703-712Crossref PubMed Scopus (67) Google Scholar, 12Rogers M. Yehiely F. Scott M. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3182-3186Crossref PubMed Scopus (158) Google Scholar, 13Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury Jr., P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (791) Google Scholar, 14Blochberger T.C. Cooper C. Peretz D. Tatzelt J. Griffith O.H. Baldwin M.A. Prusiner S.B. Protein Eng. 1997; 10: 1465-1473Crossref PubMed Scopus (38) Google Scholar). Expression of these PrP constructs was analyzed by Western blotting using the monoclonal anti-PrP antibody 3F4 (Fig. 1B). To monitor N-linked glycosylation of PrP, yeast extracts were treated with endo-β-N-acetylglucosaminidase H, which removes all asparagine-linked sugar moieties from yeast proteins (43Maley F. Trimble R.B. Tarentino A.L. Plummer Jr., T.H. Anal. Biochem. 1989; 180: 195-204Crossref PubMed Scopus (643) Google Scholar). It appeared that neither the Gas1p nor the PrP signal sequence was able to mediate the import of PrP into the yeast ER. All PrP constructs remained unglycosylated. Moreover, the majority of gPrP (∼95%), pPrPΔGPI (100%), and gPrPΔGPI (∼50%) contained uncleaved N-terminal signal sequences. In this context it is important to note that in mammalian cells PrPΔGPI is mainly unglycosylated, but in contrast to gPrPΔGPI and pPrPΔGPI expressed in yeast, it is N-terminally processed. In the same study we showed that PrP containing a nonfunctional C-terminal GPI anchor signal sequence is both N-terminally processed and core-glycosylated (10Winklhofer K.F. Heske J. Heller U. Reintjes A. Muranji W. Moarefi I. Tatzelt J. J. Biol. Chem. 2003; 278: 14961-14970Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Further biochemical analysis revealed that gPrPΔGPI (Fig. 1D), gPrP, pPrPΔGPI, and cyto-PrP (data not shown) were highly insoluble in detergent buffer and partially resistant to proteolytic digestion, corroborating earlier findings (44Ma J.Y. Lindquist S. Nat. Cell Biol. 1999; 1: 358-361Crossref PubMed Scopus (105) Google Scholar). In the course of these experiments we noticed a retarded growth of yeast cells expressing PrP with an uncleaved N-terminal signal sequence, be it gPrP, gPrPΔGPI, or pPrPΔGPI. To analyze the growth of PrP-expressing cells in more detail, yeast cultures were serially diluted and spotted onto selective agar plates (Fig. 1C, SCD-ura). Both gPrP and gPrPΔGPI showed a reduced growth compared with the vector control. Interestingly, decreased growth was specifically linked to the expression of PrP targeted to the ER and was also observed for pPrPΔGPI (data not shown). PrP expressed without a N-terminal signal peptide (cyto-PrP) had no adverse effects on cell growth. These experiments revealed that expression of mouse PrP with a signal peptide specific for post-translational targeting to the ER generates N-terminally unprocessed misfolded PrP and interferes with yeast growth. Cytosolically expressed PrP lacking the signal peptide is misfolded as well; however, this has no adverse effects on yeast growth. Deletion of the Putative Transmembrane Domain Restores Processing of PrP and Yeast Growth—Previous studies revealed that the ER import of PrP is modulated by an internal stretch of hydrophobic amino acids (aa112–135). Instead of being synthesized as a secreted protein, this domain can direct the formation of a transmembrane topology (45Lopez C.D. Yost C.S. Prusiner S.B. Myers R.M. Lingappa V.R. Science. 1990; 248: 226-229Crossref PubMed Scopus (114) Google Scholar). Different studies indicate that this stretch of hydrophobic amino acids may have additional properties; short peptides comprising residues 106–126 formed fibrils in vitro (18Tagliavini F. Prelli F. Verga L. Giaccone G. Sarma R. Gorevic P. Ghetti B. Passerini F. Ghibaudi E. Forloni G. Salmona M. Bugiani O. Frangione B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9678-9682Crossref PubMed Scopus (235) Google Scholar) and induced cell death in cultured cells (19Deli M.A. Sakaguchi S. Nakaoke R. Abraham C.S. Takahata H. Kopacek J. Shigematsu K. Katamine S. Niwa M. Neuroreport. 2000; 11: 3931-3936Crossref PubMed Scopus (37) Google Scholar, 20Brown D.R. Schmidt B. Kretzschmar H.A. Nature. 1996; 380: 345-347Crossref PubMed Scopus (499) Google Scholar, 21Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, 22Haïk S. Peyrin J.M. Lins L. Rosseneu M.Y. Brasseur R. Langeveld J.P. Tagliavini F. Deslys J.P. Lasmézas C. Dormont D. Neurobiol. Dis. 2000; 7: 644-656Crossref PubMed Scopus (42) Google Scholar). To address the role of the putative TM domain in PrP folding and ER import, several deletion mutants lacking the complete TM domain and also PrP-AV3 and PrP-G122P were included in our analysis. The triple A to V and the G to P substitutions are located within the putative transmembrane domain (Fig. 2A). Although the AV3 mutation enhances the formation of CtmPrP, the G122P mutation abolishes the formation of any transmembrane topology, be it CtmPrP or NtmPrP (17Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar, 46Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (269) Google Scholar). Western blot analysis revealed that all PrP mutants lacking the putative TM domain were glycosylated, indicated by an increased electrophoretic mobility after Endo H digestion (Fig. 2B, Endo H +). In addition, these glycosylated mutants were N-terminally processed. The mutants with amino acid substitutions in the putative TM domain, gPrP-G122P and gPrP-AV3, remained unprocessed and unglycosylated, similar to gPrP (data not shown), and interfered with yeast growth (Fig. 2C). However, cells expressing PrP mutants with a deleted putative TM domain grew at wild-type rates (Fig. 2C). Our experiments revealed that PrP mutants lacking the putative TM domain were N-terminally processed and glycosylated, although they were targeted to the ER via the Gas1p signal peptide. In addition, these mutants did not interfere with yeast growth. Unprocessed PrP Shows Prolonged Association with ER Membranes—PrP targeted to the ER via the PrP or Gas1p signal peptide (pPrP, gPrP) had biochemical properties similar to cyto-PrP and also seemed to remain in the cytosol. However, cyto-PrP did not interfere with yeast growth. To analyze the cellular localization of the different PrP mutants in more detail, we performed a Renografin density gradient centrifugation (39Schandel K.A. Jenness D.D. Mol. Cell. Biol. 1994; 14: 7245-7255Crossref PubMed Google Scholar, 40Wang Q. Chang A. EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar). This analysis revealed a specific feature of PrP mutants that interfered with yeast growth. As expected, cytosolic PrP remained in the bottom fractions together with the subunit Gim2p (34Geissler S. Siegers K. Schiebel E. EMBO J. 1998; 17: 952-966Crossref PubMed Scopus (236) Google Scholar) of the cytosolic GimC complex (Fig. 3A). The glycosylated and N-terminally processed PrP mutant with a deleted TM domain (gPrPΔ28–156) was found in the same fractions as Vhp1p, the 100-kDa subunit of the vacuolar V-ATPase (47Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T.H. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar). Further analysis revealed that the other N-terminally processed and glycosylated PrP mutants analyzed, gPrPΔTM and gPrPΔ28–140, were also exported from the ER and transported to the vacuole (data not shown). In contrast, the N-terminally unprocessed PrP mutant gPrPΔGPI, as well as gPrP, gPrP-AV3, and gPrP-G122P (data not shown), was membrane-associated and co-localized with the ER and plasma membrane fraction. Because gPrPΔGPI has no GPI anchor and was not secreted into the culture medium (data not shown), we assumed that it was associated with ER vesicles. In summary, these data indicate that the PrP mutants characterized by an uncleaved N-terminal signal peptide have a unique feature; they show prolonged association with ER membranes. In Mammalian Cells PrPΔGPI Is N-terminally Processed and Transported through the Secretory Pathway—Our analysis in yeast revealed that PrPΔGPI expression significantly interfered with cell growth. The question arose whether a similar phenotype could be observed in mammalian cells. PrPΔGPI was previously characterized by us and other groups, and adverse effects on the growth of mammalian cells have not been described (10Winklhofer K.F. Heske J. Heller U. Reintjes A. Muranji W. Moarefi I. Tatzelt J. J. Biol. Chem. 2003; 278: 14961-14970Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11Walmsley A.R. Zeng F.N. Hooper N.M. EMBO J. 2001; 20: 703-712Crossref PubMed Scopus (67) Go" @default.
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• W2017014906 title "Post-translational Import of the Prion Protein into the Endoplasmic Reticulum Interferes with Cell Viability" @default.
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