Signal peptide peptidase dependent cleavage of type II transmembrane substrates releases intracellular and extracellular signals
Kumlesh K. DevSandipan ChatterjeeMaribel OsindeDaniela StaufferHannah L. MorganMonika KobialkoUwe DenglerHeinrich RueegerBruno MartoglioGiorgio Rovelli
29
Citation
28
Reference
10
Related Paper
Citation Trend
Keywords:
Cleavage (geology)
Signal peptidase
The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases. The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases. Prion diseases are fatal neurological disorders of humans and animals that appear in sporadic, familial, and infectiously acquired forms. These disorders are caused by conversion of a normal neuronal glycoprotein (PrPC) 1The abbreviations used are: PrPC, cellular isoform of PrP; PrPSc, scrapie isoform of PrP; CHO, Chinese hamster ovary; GPI, glycosylphosphatidylinositol; PK, proteinase K; PrP, prion protein; SP, signal peptide; WT, wild type; ER, endoplasmic reticulum; PNGase, peptide:N-glycosidase.1The abbreviations used are: PrPC, cellular isoform of PrP; PrPSc, scrapie isoform of PrP; CHO, Chinese hamster ovary; GPI, glycosylphosphatidylinositol; PK, proteinase K; PrP, prion protein; SP, signal peptide; WT, wild type; ER, endoplasmic reticulum; PNGase, peptide:N-glycosidase. into a conformationally altered isoform (PrPSc) that is infectious in the absence of nucleic acid (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5130) Google Scholar, 2Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1108) Google Scholar). PrPC, which is soluble and protease-sensitive, consists of an α-helical, C-terminal domain and an unstructured N-terminal domain. In contrast, PrPSc is rich in β-sheets, aggregated, and protease-resistant. The physiological function of PrPC is uncertain but may be related to transport of copper ions or protection from oxidative stress (3Brown L.R. Harris D.A. Massaro E.J. Handbook of Copper Pharmacology and Toxicology. Humana Press Inc., Totowa, NJ2002: 103-113Google Scholar). PrPC is unusual because it can adopt multiple membrane topologies. Most PrPC molecules are attached to the outer leaflet of the plasma membrane through a C-terminal glycosylphosphatidylinositol (GPI) anchor (this topology is designated SecPrP) (4Stahl N. Borchelt D.R. Prusiner S.B. Biochemistry. 1990; 29: 5405-5412Crossref PubMed Scopus (225) Google Scholar, 5Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). However, some PrPC molecules assume a transmembrane orientation when synthesized in vitro or in cells (6De Fea K.A. Nakahara D.H. Calayag M.C. Yost C.S. Mirels L.F. Prusiner S.B. Lingappa V.R. J. Biol. Chem. 1994; 269: 16810-16820Abstract Full Text PDF PubMed Google Scholar, 7Hegde 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, 8Hölscher C. Bach U.C. Dobberstein B. J. Biol. Chem. 2001; 276: 13388-13394Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 9Kim S.J. Rahbar R. Hegde R.S. J. Biol. Chem. 2001; 276: 26132-26140Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Rutkowski D.T. Lingappa V.R. Hegde R.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7823-7828Crossref PubMed Scopus (62) Google Scholar, 11Kim S.J. Hegde R.S. Mol. Biol. Cell. 2002; 13: 3775-3786Crossref PubMed Scopus (66) Google Scholar). These forms, designated NtmPrP and CtmPrP, span the lipid bilayer once via a highly conserved hydrophobic region in the center of the molecule (amino acids 111-134), with either the N or C terminus, respectively, on the extracytoplasmic side of the membrane. It has been shown that these species are generated in small amounts (<10% of the total PrP) as part of the normal biosynthesis of wild-type PrP in the endoplasmic reticulum (ER). However, mutations within or near the transmembrane domain, including an A117V mutation linked to GSS as well as several "artificial" mutations not seen in human patients, increase the relative proportion of CtmPrP to as much as 20-30% of the total (7Hegde 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, 12Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (268) Google Scholar). Recent studies have begun to define the mechanisms responsible for determining PrP topology during the translation process. We discovered that a non-conservative substitution (L9R) within the hydrophobic core of the signal sequence dramatically increased the proportion of CtmPrP (13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar). Combining this mutation with a triple substitution (3AV) within the transmembrane domain resulted in a molecule that was synthesized exclusively as CtmPrP. These results indicated that the signal sequence as well as the transmembrane domain were major determinants of PrP topology. Work by Hegde and colleagues (9Kim S.J. Rahbar R. Hegde R.S. J. Biol. Chem. 2001; 276: 26132-26140Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Rutkowski D.T. Lingappa V.R. Hegde R.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7823-7828Crossref PubMed Scopus (62) Google Scholar, 11Kim S.J. Hegde R.S. Mol. Biol. Cell. 2002; 13: 3775-3786Crossref PubMed Scopus (66) Google Scholar) has demonstrated that these two determinants act in mechanistically distinct ways. The signal sequence serves a dual function, first targeting the nascent polypeptide chain to the translocon channel in the ER membrane via binding to the signal recognition particle, and subsequently gating the translocon to allow passage of the N terminus into the ER lumen. In contrast, the transmembrane domain acts primarily to trigger integration of the polypeptide into the lipid bilayer. The combined action of both domains operating during the translocation process serves to regulate the proportions of the three topological variants of PrP. Regulatory factors associated with the translocon, in addition to sequence determinants within the PrP molecule itself, have also been shown to influence the final topology achieved (14Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 2: 85-91Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 15Fons R.D. Bogert B.A. Hegde R.S. J. Cell Biol. 2003; 160: 529-539Crossref PubMed Scopus (196) Google Scholar). Work from our laboratory has identified several novel cell biological features of CtmPrP. First, CtmPrP contains an uncleaved, N-terminal signal peptide (13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar). This characteristic makes CtmPrP unusual among other type II transmembrane proteins, most of which have internal signal-anchor sequences. Second, CtmPrP has a C-terminal GPI anchor in addition to a transmembrane domain, thus displaying an unusual, dual mode of membrane attachment (13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar, 16Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Finally, by using the L9R/3AV mutant, we found that CtmPrP expressed in cultured cells remains core-glycosylated and is retained completely in the ER (13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar). This result implies that the protein is recognized as abnormal by the ER quality control machinery that monitors folding of newly synthesized polypeptides. A great deal of interest in the subject of PrP membrane topology derives from the possibility that topological variants of PrP may play an important pathogenic role in prion diseases. Although PrPSc is widely agreed to be the infectious form of PrP, there is considerable debate about whether it is the form responsible for neuronal loss in these disorders (17Chiesa R. Harris D.A. Neurobiol. Dis. 2001; 8: 743-763Crossref PubMed Scopus (146) Google Scholar). The amount, anatomical distribution, and time course of accumulation of PrPSc often correlates with the development of neuropathology and clinical symptoms, but there are notable exceptions to this association. These discrepancies have led to the hypothesis that alternate forms of PrP, distinct from both PrPC and PrPSc, are the proximate causes of neurodegeneration. One candidate for such a neurotoxic intermediate is CtmPrP. Two major pieces of evidence have been used to argue that CtmPrP plays a key pathogenic role. First, transgenic mice have been generated that synthesize PrP molecules carrying the A117V mutation or one of the other CtmPrP-favoring mutations (7Hegde 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, 12Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (268) Google Scholar). Animals expressing the mutant proteins above a threshold level synthesize CtmPrP in their brains and spontaneously develop a scrapie-like neurological illness, but without PrPSc detectable by Western blotting or infectivity assays. This result implies that certain familial forms of PrP may be due directly to increased levels of CtmPrP. Second, mice have been constructed in which a wild-type hamster PrP transgene serves as a reporter of CtmPrP formation (12Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (268) Google Scholar). When these animals are inoculated with mouse prions, the amounts of CtmPrP as well as PrPSc in the brain are found to increase during the course of the infection. This result has been interpreted to indicate that PrPSc induces formation of CtmPrP, which is then the proximate cause of neurodegeneration during infectiously acquired prion diseases. In this view then, CtmPrP is a key intermediate in both genetic and infectious prion diseases. Another topological variant of PrP that has been proposed as a neurotoxic intermediate is cytosolic PrP. Expression of an artificial form of PrP lacking a signal sequence, which presumably favors accumulation of PrP in the cytoplasm, has been found to be toxic to cultured cells and transgenic mice (18Ma J. Wollmann R. Lindquist S. Science. 2002; 298: 1781-1785Crossref PubMed Scopus (429) Google Scholar). However, there is debate about whether PrP is found in the cytoplasm under normal circumstances and, if so, what mechanisms are responsible for delivering it there. Based on the observation that cytosolic PrP accumulates in cells that have been treated with proteasome inhibitors, it has been suggested that some molecules are retrotranslocated into the cytoplasm from the ER lumen as part of normal ER quality control mechanisms (19Ma J. Lindquist S. Science. 2002; 298: 1785-1788Crossref PubMed Scopus (268) Google Scholar, 20Ma J. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14955-14960Crossref PubMed Scopus (268) Google Scholar, 21Yedidia Y. Horonchik L. Tzaban S. Yanai A. Taraboulos A. EMBO J. 2001; 20: 5383-5391Crossref PubMed Scopus (223) Google Scholar). In contrast, our experiments indicate that cytosolic PrP molecules represents untranslocated chains that have never entered the ER (22Drisaldi B. Stewart R.S. Adles C. Stewart L.R. Quaglio E. Biasini E. Fioriti L. Chiesa R. Harris D.A. J. Biol. Chem. 2003; 278: 21732-21743Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). These chains, which are observed primarily under conditions of protein overexpression, contain an uncleaved N-terminal signal peptide and lack a GPI anchor. A key gap in the experimental evidence supporting roles for CtmPrP and cytosolic PrP in prion-induced neurodegeneration is the lack of data demonstrating that the amounts of these forms increase during the course of a prion infection. In part, this difficulty is due to the absence of direct methods for detecting CtmPrP and cytosolic PrP in infected cells and tissues. In this paper, we present characterization of an antibody that reacts with both CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides and our use of this antibody to assay CtmPrP and cytosolic PrP in infected samples. In addition, we carry out a mutational analysis of several sequence determinants in PrP to better understand the factors that influence the topology of the protein. Plasmids—Synthetic oligonucleotides encoding point mutations in the PrP signal sequence (Fig. 1A) were used to amplify a portion of the PrP DNA sequence by PCR. DNA fragments carrying the mutation were digested with HindIII and PshA1 and cloned into a pcDNA3 plasmid (Invitrogen) containing the WT mouse PrP sequence from which the HindIII-PshA1 fragment had been removed. Plasmids encoding PrP molecules with a FLAG epitope (DYKDDDDK) inserted at position 22/23, and with altered numbers of octapeptide repeats (PG0, Δ51-90; PG1, Δ51-82; PG2, Δ67-90; PG14, +9 repeats) have been described previously (5Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar, 23Quaglio E. Chiesa R. Harris D.A. J. Biol. Chem. 2001; 276: 11432-11438Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Other PrP mutants were constructed by PCR (16Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). All PrP coding regions carried an epitope tag for monoclonal antibody 3F4, created by changing residues 108 and 111 to methionine. Prior to in vitro transcription, plasmids were linearized with XbaI. In Vitro Translation and PK Protection—mRNAs encoding WT and mutant PrP molecules were transcribed using the mMessage mMachine kit (Ambion, Austin, TX) and were translated using rabbit reticulocyte lysate (Promega, Madison, WI) containing [35S]methionine as directed by the manufacturer, except that the final lysate concentration was 50%. Translation reactions were supplemented with microsomal membranes from mouse BW5174.3 cells (24Vidugiriene J. Menon A.K. EMBO J. 1995; 14: 4686-4694Crossref PubMed Scopus (25) Google Scholar) or from canine pancreas (Promega). After translation, 5-μl aliquots of lysate were incubated for 60 min at 4 °C in a final volume of 50 μl with or without 100 μg/ml PK (Roche Applied Science) in the presence or absence of 0.5% Triton X-100. PK was inactivated with phenylmethylsulfonyl fluoride for 5 min, and 12-μl aliquots were added to gel sample buffer containing phenylmethylsulfonyl fluoride for analysis by SDS-PAGE. In some cases, PrP was immunoprecipitated from translation reactions (as described below) prior to SDS-PAGE. For enzymatic deglycosylation, PrP was eluted from protein A-Sepharose beads with 1% SDS, 50 mm Tris-HCl (pH 7.5) and was then diluted 10-fold with 50 mm Tris-HCl (pH 7.5), 0.5% Triton X-100 containing 0.33 units/ml N-glycosidase F (New England Biolabs). After incubation at 37 °C for 1 h, proteins were precipitated with methanol and analyzed by SDS-PAGE. Radioactive bands on gels were quantitated using a PhosphorImager SI (Amersham Biosciences). Scrapie Infection of N2a Cells—Highly scrapie-susceptible sub-clones of N2a cells were prepared as described (25Bosque P.J. Prusiner S.B. J. Virol. 2000; 74: 4377-4386Crossref PubMed Scopus (185) Google Scholar). Briefly, N2a cells from the ATCC (CCL131) were first sub-cloned by limiting dilution. Each sub-clone was then tested for scrapie susceptibility by incubation for 3 days with an extract of N2a cells that had been infected previously with the Chandler strain of scrapie (26Nishida N. Harris D.A. Vilette D. Laude H. Frobert Y. Grassi J. Casanova D. Milhavet O. Lehmann S. J. Virol. 2000; 74: 320-325Crossref PubMed Scopus (215) Google Scholar). Cells were then passaged for 6 weeks and analyzed for PrP 27-30 by cell blotting or by Western blotting after PK digestion. The susceptible sub-clone used for the experiment shown in Fig. 7 was designated N2a.3. It was used in the infected state, as well as in the uninfected state as a matched control. Transfection, Metabolic Labeling, and Immunoprecipitation—CHO and N2a cells were transiently transfected with PrP-encoding plasmids using LipofectAMINE or LipofectAMINE 2000 (Invitrogen) according to the manufacturer's directions. Twenty-four hours after transfection, cells were labeled for 6 h in methionine- and cysteine-free medium containing 100-200 μCi/ml of [35S]methionine/cysteine (Promix; Amersham Biosciences). Cultures were then lysed in 0.5% SDS, 50 mm Tris-HCl (pH 7.5), heated at 95 °C for 5 min, and diluted with 10 volumes of RIPA buffer (150 mm NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mm Tris-HCl (pH 7.5)). Diluted lysates were incubated with anti-PrP antiserum for > 1 h at 4 °C and then with 20 μl of protein A-Sepharose beads for 30 min at 4 °C. Beads were washed four times in RIPA buffer, and PrP was eluted by heating at 95 °C in gel sample buffer. Scrapie Infection of Mice and Hamsters—Tg(WT)/Prn-p 0/0 mice (E1 line) expressing wild-type PrP carrying a 3F4 epitope have been described previously (27Chiesa R. Piccardo P. Ghetti B. Harris D.A. Neuron. 1998; 21: 1339-1351Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Tg(L9R/3AV)/Prn-p 0/0 mice (B line) express mouse PrP carrying an L9R/3AV mutation and a 3F4 tag, a construct we have expressed previously in cultured cells (13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar). Full characterization of these mice will be provided elsewhere. 2R. S. Stewart and D. A. Harris, manuscript in preparation. Scrapie inocula included the hamster 263K strain, the mouse RML strain, and the RML strain that had been passaged once in Tg(WT) mice to introduce the 3F4 epitope. To prepare inocula, infected brains were homogenized (10%, w/v) in phosphate-buffered saline using sterile, disposable tissue grinders. After clearing by centrifugation at 900 × g for 5 min, the homogenates were diluted to a final concentration of 1 or 2.5% in PBS, and 25 μl was injected intracerebrally into the right parietal lobe of 4-6- week-old recipient mice or hamsters using a 25-gauge needle. Western Blots of Brain Homogenates—Brain lysates were prepared in 0.5% SDS, 50 mm Tris-HCl (pH 7.5). Samples were heated at 95 °C for 5 min and diluted 10-fold with 50 mm Tris-HCl (pH 7.5), 0.5% Triton X-100 containing 0.33 units/ml N-glycosidase F. After incubation at 37 °C for 2 h, proteins were precipitated with methanol, separated by SDS-PAGE, and subjected to Western blotting with 3F4 antibody. Antibodies—An antibody (anti-SP) that selectively recognizes forms of murine PrP containing an uncleaved signal peptide was generated by immunizing rabbits with a synthetic peptide (TMWTDVGLCKKRPK; amino acids 14-27) that spans the signal peptide cleavage site at residues 22/23. The peptide was conjugated to keyhole limpet hemocyanin using both glutaraldehyde and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester (Sigma). Monoclonal antibodies 3F4 (28Kascsak R.J. Rubinstein R. Merz P.A. Tonna-DeMasi M. Fersko R. Carp R.I. Wisniewski H.M. Diringer H. J. Virol. 1987; 61: 3688-3693Crossref PubMed Google Scholar) and 8H4 (29Zanusso G. Liu D. Ferrari S. Hegyi I. Yin X. Aguzzi A. Hornemann S. Liemann S. Glockshuber R. Manson J.C. Brown P. Petersen R.B. Gambetti P. Sy M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8812-8816Crossref PubMed Scopus (175) Google Scholar) and polyclonal antibody P45-66 (5Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) against PrP have been described previously. Mutations in the Hydrophobic Core of the Signal Peptide Increase the Proportion of CtmPrP and Reduce Translocation Efficiency—For these experiments, we translated PrP mRNA in the presence of microsomes from murine thymoma cells, which are efficient at adding the C-terminal GPI anchor (16Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Translation reactions were subsequently subjected to PK digestion to reveal protease-protected products corresponding to SecPrP and CtmPrP. NtmPrP was not quantitated in these experiments, because negligible amounts of this form are produced in the presence of thymoma microsomes (16Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We showed previously that substitution of arginine for leucine at position 9 (L9R) in the hydrophobic core (h-region) of the signal peptide had a dramatic effect on PrP membrane topology, with ∼50% of the translocated protein assuming the CtmPrP orientation, compared with ∼10% for WT PrP (13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar). We then tested the effects of other amino acid substitutions at this site. The results are shown in Fig. 1 and summarized in Table I (lines 1-12). Substitution of either positively charged residues (Arg and Lys) or a negatively charged residue (Asp) for leucine at position 9 increased the proportion of CtmPrP, with two non-polar residues (Pro and Gly) having very little effect. All of the substitutions also significantly reduced the efficiency of translocation (the total percentage of PK-protected chains) from ∼25% for WT PrP to 5-15% for the mutants (data not shown). To examine the effect of substitutions at another residue in the h-region of the signal peptide, we analyzed V13R and V13D. Both of these mutations completely abolished translocation (Fig. 1).Table IQuantitation of Ctm Prp produced by the mutants used in this studyConstructCtmPrP%1. WT12.42. L9R30.13. L9R/3AV87.64. L9K62.65. L9K/3AV87.76. L9D43.07. L9D/3AV77.58. L9G22.39. L9G/3AV67.810. L9P11.011. V13RaNeither CtmPrP nor SecPrP were present at detectable levels.12. V13DaNeither CtmPrP nor SecPrP were present at detectable levels.13. WT11.814. G20W7.415. C22Y13.716. C22Y/L9R/3AV90.017. WT 23-254aNeither CtmPrP nor SecPrP were present at detectable levels.18. 3AV 23-254aNeither CtmPrP nor SecPrP were present at detectable levels.19. WT31.320. FLAG/WT58.721. 3AV54.322. FLAG/3AV89.623. PG021.324. PG114.725. PG214.326. PG5(WT)11.227. PG145.528. 3AV27.029. WT/GPI+25.330. WT/GPI−24.931. 3AV46.132. 3AV/GPI−39.5a Neither CtmPrP nor SecPrP were present at detectable levels. Open table in a new tab Previous work demonstrated that certain mutations in the transmembrane segment also increased the amount of CtmPrP. One mutation that has been studied extensively is the triple substitution designated 3AV (substitution of valine for alanine at positions 112, 114, and 117) (7Hegde 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, 13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar). When this mutation was combined with mutations in the signal sequence, an additive increase in the percentage of CtmPrP was observed (Fig. 1; Table I, lines 1-12). PrP molecules carrying the 3AV mutation along with substitution of a charged amino acid (Arg, Lys, and Asp) at position 9 were synthesized almost exclusively as CtmPrP. Lack of Signal Peptide Cleavage Does Not Cause Production of CtmPrP—We have shown previously (13Stewart R.S. Drisaldi B. Harris D.A. Mol. Biol. Cell. 2001; 12: 881-889Crossref PubMed Scopus (114) Google Scholar) that CtmPrP has an uncleaved signal peptide. However, it remained unknown whether the lack of signal peptide cleavage was a cause or a consequence of CtmPrP formation. To address this question, we introduced mutations that prevent cleavage by signal peptidase, and we assayed their effect on synthesis of CtmPrP. The -1 and -3 positions in the c-region of signal peptides have been shown to have the greatest influence on cleavage (30von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (858) Google Scholar); small polar residues are strongly preferred. We therefore substituted large, hydrophobic residues at these positions to block the action of signal peptidase (Fig. 2A). Fig. 2B (lanes 2, 5, and 8) shows that these mutations had no effect on PrP topology. As in the WT molecule, PrP containing either G20W or C22Y substitutions produced primarily SecPrP, with ∼10% being synthesized as CtmPrP (Table I, lines 13-15). This result was verified by assaying the topology of these mutants in transfected cells (data not shown). The mutant C22Y/L9R/3AV produced 90% CtmPrP (Fig. 2B, lane 11; Table I, line 16), indicating that lack of signal peptide cleavage does not inhibit formation of CtmPrP. We confirmed that the signal peptide remained uncleaved in the G20W and C22Y mutants by observing the slightly reduced mobility of the SecPrP bands on SDS-PAGE (Fig. 2B, compare lane 2 to lanes 5 and 8). We conclude from these results that lack of signal peptide cleavage is a consequence, and not a cause, of CtmPrP formation. Deletion of the Signal Peptide Prevents Translocation— CtmPrP has the topology of a type II transmembrane protein (N terminus in the cytoplasm). Most proteins of this type contain an internal signal-anchor sequence that initiates translocation by binding to the signal recognition particle, and also anchors the polypeptide chain in the lipid bilayer. To test whether the transmembrane segment of PrP could serve a membrane targeting function independent of the N-terminal signal sequence, we assayed PrP constructs in which the N-terminal sequence had been deleted. We observed that removal of the signal peptide completely abolished tr
Cite
Citations (80)
Helix (gastropod)
Alpha helix
Cite
Citations (20)
Signal peptidase
Cleavage (geology)
Cite
Citations (7)
The structural proteins of rubella virus (RV) are translated as a large polyprotein precursor, p110, which is processed to produce the mature virion components, the 33K capsid protein (C) and the two envelope glycoproteins, E1 (58K) and E2 (42K to 47K). The precise processing mechanism has not been elucidated; however it must include at least two proteolytic cleavages to release the individual virion components from the polyprotein, and it must provide for their dichotomous intracellular distribution. The C protein remains in the cytoplasm where it participates in the formation of nucleocapsids, while the envelope glycoproteins enter the cellular secretory pathway and are N-glycosylated and cleaved. Sequence analysis of the 24S mRNA encoding the polyprotein precursor suggests that both E1 and E2 are preceded by signal peptides for translocation across the membrane of the rough endoplasmic reticulum. A recent study has provided direct evidence that the putative signal peptide preceding E1 can in fact mediate translocation of E1. In this study, we have used in vitro translation- translocation assays to examine further the processing of RV glycoproteins. We have shown that the putative signal sequence preceding E2 can mediate translocation of the E2 protein in the absence of an intact E1 signal peptide. The experiments also revealed that cleavage of the E2-E1 polyprotein requires (i) the E2 signal peptide, (ii) microsomal membranes and (iii) sequences beyond the proximal half of the E1 signal peptide. Together these results suggest that separation of the E2 signal sequence as well as the proteolytic cleavage of El from E2 is performed by the cellular enzyme, signal peptidase.
Signal peptidase
Cleavage (geology)
Rubella virus
Cite
Citations (14)
Cite
Citations (0)
Signal peptidase
Chitosanase
Streptomycetaceae
Cite
Citations (12)
Signal peptidase
Signal recognition particle
Protein Sorting Signals
Cite
Citations (7)
Toll-like receptors (TLRs) act as the first line of defense against bacterial and viral pathogens by initiating critical defense signals upon dimer activation. The contribution of the transmembrane domain in the dimerization and signaling process has heretofore been overlooked in favor of the extracellular and intracellular domains. As mounting evidence suggests that the transmembrane domain is a critical region in several protein families, we hypothesized that this was also the case for Toll-like receptors. Using a combined biochemical and biophysical approach, we investigated the ability of isolated Toll-like receptor transmembrane domains to interact independently of extracellular domain dimerization. Our results showed that the transmembrane domains had a preference for the native dimer partners in bacterial membranes for the entire receptor family. All TLR transmembrane domains showed strong homotypic interaction potential. The TLR2 transmembrane domain demonstrated strong heterotypic interactions in bacterial membranes with its known interaction partners, TLR1 and TLR6, as well as with a proposed interaction partner, TLR10, but not with TLR4, TLR5, or unrelated transmembrane receptors providing evidence for the specificity of TLR2 transmembrane domain interactions. Peptides for the transmembrane domains of TLR1, TLR2, and TLR6 were synthesized to further study this subfamily of receptors. These peptides validated the heterotypic interactions seen in bacterial membranes and demonstrated that the TLR2 transmembrane domain had moderately strong interactions with both TLR1 and TLR6. Combined, these results suggest a role for the transmembrane domain in Toll-like receptor oligomerization and as such, may be a novel target for further investigation of new therapeutic treatments of Toll-like receptor mediated diseases.
Cite
Citations (75)
The homotrimeric SARS-CoV-2 spike protein enables viral infection by mediating the fusion of the viral envelope with the host membrane. The spike protein is anchored to the SARS-CoV-2 envelope by its transmembrane domain (TMD), which is composed of three TM helices, each contributed by one of the protomers of the homotrimeric spike. Although the TMD is important for SARS-CoV-2 viral fusion and is well-conserved across the Coronaviridae family, it is unclear whether it is a passive anchor of the spike or actively promotes viral fusion. Specifically, the nature of the TMD dynamics and how these dynamics couple to the large pre- to post-fusion conformational transition of the spike ectomembrane domains remains unknown. Here, we computationally study the SARS-CoV-2 spike TMD in both homogenous POPC and cholesterol containing membranes to characterize its structure, dynamics, and self-assembly. Different tools identify distinct segments of the spike sequence as its TM helix. Atomistic simulations of a spike protomer segment that includes the superset of the TM helix predictions show that the membrane-embedded TM sequence bobs, tilts and gains and loses helicity at the membrane edges. Coarse-grained multimerization simulations using representative TM helix structures from the atomistic simulations exhibit diverse trimer populations whose architecture depends on the structure of the TM helix protomer. Multiple overlapping and conflicting dimerization interfaces stabilized these trimeric populations. An asymmetric conformation is populated in addition to a symmetric conformation and several in-between trimeric conformations. While the symmetric conformation reflects the symmetry of the resting spike, the asymmetric TMD conformation could promote viral membrane fusion through the stabilization of a fusion intermediate. Together, our simulations demonstrate that the SARS-CoV-2 spike TM anchor sequence is inherently dynamic, trimerization does not abrogate these dynamics and the various observed TMD conformations may enable viral fusion.
Helix (gastropod)
Sequence (biology)
Trimer
Cite
Citations (1)