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• W2038049042 abstract "N-terminal signal sequences mediate endoplasmic reticulum (ER) targeting and insertion of nascent secretory and membrane proteins and are, in most cases, cleaved off by signal peptidase. The mouse mammary tumor virus envelope protein and its alternative splice variant Rem have an unusually long signal sequence, which contains a nuclear localization signal. Although the envelope protein is targeted to the ER, inserted, and glycosylated, Rem has been described as a nuclear protein. Rem as well as a truncated version identical to the cleaved signal sequence have been shown to function as nuclear export factors for intron-containing transcripts. Using transiently transfected cells, we found that Rem is targeted to the ER, where the C-terminal portion is translocated and glycosylated. The signal sequence is cleaved off and accumulates in nucleoli. In a cell-free in vitro system, the generation of the Rem signal peptide depends on the presence of microsomal membranes. In vitro and in cells, the signal peptide initially accumulates in the membrane and is subsequently released into the cytosol. This release does not depend on processing by signal peptide peptidase, an intramembrane cleaving protease that can mediate the liberation of signal peptide fragments from the ER membrane. Our study suggests a novel pathway by which a signal peptide can be released from the ER membrane to fulfill a post-targeting function in a different compartment. N-terminal signal sequences mediate endoplasmic reticulum (ER) targeting and insertion of nascent secretory and membrane proteins and are, in most cases, cleaved off by signal peptidase. The mouse mammary tumor virus envelope protein and its alternative splice variant Rem have an unusually long signal sequence, which contains a nuclear localization signal. Although the envelope protein is targeted to the ER, inserted, and glycosylated, Rem has been described as a nuclear protein. Rem as well as a truncated version identical to the cleaved signal sequence have been shown to function as nuclear export factors for intron-containing transcripts. Using transiently transfected cells, we found that Rem is targeted to the ER, where the C-terminal portion is translocated and glycosylated. The signal sequence is cleaved off and accumulates in nucleoli. In a cell-free in vitro system, the generation of the Rem signal peptide depends on the presence of microsomal membranes. In vitro and in cells, the signal peptide initially accumulates in the membrane and is subsequently released into the cytosol. This release does not depend on processing by signal peptide peptidase, an intramembrane cleaving protease that can mediate the liberation of signal peptide fragments from the ER membrane. Our study suggests a novel pathway by which a signal peptide can be released from the ER membrane to fulfill a post-targeting function in a different compartment. Signal sequences are N-terminal extensions on nascent secretory and membrane proteins and mediate translocation across or insertion into the membrane of the endoplasmic reticulum. They typically include 15–25 amino acid residues and have a tripartite structure with a hydrophobic core region flanked by a positively charged n-region and a c-region. The latter includes the signal peptidase cleavage site. Cleaved signal sequences, named signal peptides, are thought to be degraded, but some accumulate or are further processed by an intramembrane cleaving protease named signal peptide peptidase (SPP) 5The abbreviations used are:SPPsignal peptide peptidaseMMTVmouse mammary tumor virusEnvenvelope proteingpglycoproteinNLSnuclear localization signalRemregulator of export/expression of MMTVGAPDHglyceraldehyde-3-phosphate dehydrogenaseRMsrough microsomesPrlprolactinHERV-Khuman endogenous retrovirus KERendoplasmic reticulumEGFPenhanced green fluorescent proteinTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePBSphosphate-buffered saline. (1Martoglio B. Dobberstein B. Trends Cell Biol. 1998; 8: 410-415Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 2Hegde R.S. Bernstein H.D. Trends Biochem. Sci. 2006; 31: 563-571Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Signal peptides or signal peptide fragments can have a function beyond targeting. For example, the signal peptides of several arenaviral (Lassa, Junín, and lymphocytic choriomeningitis virus) glycoproteins remain membrane-inserted. They are necessary for glycoprotein processing, part of the mature glycoprotein complexes, and important for viral infection (3York J. Romanowski V. Lu M. Nunberg J.H. J. Virol. 2004; 78: 10783-10792Crossref PubMed Scopus (129) Google Scholar, 4Agnihothram S.S. York J. Nunberg J.H. J. Virol. 2006; 80: 5189-5198Crossref PubMed Scopus (68) Google Scholar, 5York J. Nunberg J.H. J. Virol. 2006; 80: 7775-7780Crossref PubMed Scopus (103) Google Scholar, 6Eichler R. Lenz O. Strecker T. Eickmann M. Klenk H.D. Garten W. EMBO Rep. 2003; 4: 1084-1088Crossref PubMed Scopus (121) Google Scholar, 7Eichler R. Lenz O. Strecker T. Garten W. FEBS Lett. 2003; 538: 203-206Crossref PubMed Scopus (83) Google Scholar, 8Froeschke M. Basler M. Groettrup M. Dobberstein B. J. Biol. Chem. 2003; 278: 41914-41920Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 9Schrempf S. Froeschke M. Giroglou T. von Laer D. Dobberstein B. J. Virol. 2007; 81: 12515-12524Crossref PubMed Scopus (31) Google Scholar). The signal peptide of prolactin (10Weihofen A. Lemberg M.K. Ploegh H.L. Bogyo M. Martoglio B. J. Biol. Chem. 2000; 275: 30951-30956Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 11Lyko F. Martoglio B. Jungnickel B. Rapoport T.A. Dobberstein B. J. Biol. Chem. 1995; 270: 19873-19878Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), the HLA-A*0301 molecule (12Lemberg M.K. Bland F.A. Weihofen A. Braud V.M. Martoglio B. J. Immunol. 2001; 167: 6441-6446Crossref PubMed Scopus (153) Google Scholar), and the internal signal sequences of the hepatitis C virus polyprotein (13McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (394) Google Scholar) are processed by SPP, which results in the liberation of signal peptide fragments into the cytosol. The HLA-A*0301-derived signal peptide fragments are presented at the cell surface and monitor the expression of their corresponding protein for immunosurveillance (12Lemberg M.K. Bland F.A. Weihofen A. Braud V.M. Martoglio B. J. Immunol. 2001; 167: 6441-6446Crossref PubMed Scopus (153) Google Scholar). For the hepatitis C virus polyprotein, SPP processing results in the release of the core protein into the cytosol (13McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (394) Google Scholar) and affects the formation of virus-like particles (14Majeau N. Gagne V. Bolduc M. Leclerc D. J. Gen. Virol. 2005; 86: 3055-3064Crossref PubMed Scopus (17) Google Scholar, 15Ait-Goughoulte M. Hourioux C. Patient R. Trassard S. Brand D. Roingeard P. J. Gen. Virol. 2006; 87: 855-860Crossref PubMed Scopus (52) Google Scholar, 16Vauloup-Fellous C. Pene V. Garaud-Aunis J. Harper F. Bardin S. Suire Y. Pichard E. Schmitt A. Sogni P. Pierron G. Briand P. Rosenberg A.R. J. Biol. Chem. 2006; 281: 27679-27692Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). signal peptide peptidase mouse mammary tumor virus envelope protein glycoprotein nuclear localization signal regulator of export/expression of MMTV glyceraldehyde-3-phosphate dehydrogenase rough microsomes prolactin human endogenous retrovirus K endoplasmic reticulum enhanced green fluorescent protein N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phosphate-buffered saline. The mouse mammary tumor virus (MMTV), a type B retrovirus, is a causative agent of murine mammary carcinomas and is also associated with T-cell lymphomas (17Michalides R. Wagenaar E. Hilkens J. Hilgers J. Groner B. Hynes N.E. J. Virol. 1982; 43: 819-829Crossref PubMed Google Scholar, 18Racevskis J. Sarkar N.H. J. Virol. 1982; 42: 804-813Crossref PubMed Google Scholar). Its envelope protein (Env) is synthesized at the ER as a 73-kDa precursor, the signal sequence is cleaved, and the protein is further processed into the two glycoproteins, gp52 and gp36 (19Dickson C. Atterwill M. J. Virol. 1980; 35: 349-361Crossref PubMed Google Scholar, 20Redmond S.M. Dickson C. EMBO J. 1983; 2: 125-131Crossref PubMed Scopus (49) Google Scholar). The Env signal sequence is predicted to comprise 98 amino acid residues and includes a consensus sequence for a nuclear localization signal (NLS, Fig. 1A) in its extended n-region (21Hoch-Marchaim H. Weiss A.M. Bar-Sinai A. Fromer M. Adermann K. Hochman J. Virology. 2003; 313: 22-32Crossref PubMed Scopus (16) Google Scholar). Recently, an alternative splice variant of the env mRNA was identified. The translation product, named regulator of export/expression of MMTV mRNA (Rem), shares identity with Env in the predicted signal sequence, as well as the N-terminal 162 and the C-terminal 41 amino acid residues of Env (Fig. 1A) (22Indik S. Gunzburg W.H. Salmons B. Rouault F. Virology. 2005; 337: 1-6Crossref PubMed Scopus (85) Google Scholar, 23Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (102) Google Scholar). Rem was detected as a 39-kDa protein in mouse mammary tumor cells (GR) and CrFK cells stably transfected with a complete MMTV provirus (22Indik S. Gunzburg W.H. Salmons B. Rouault F. Virology. 2005; 337: 1-6Crossref PubMed Scopus (85) Google Scholar), whereas Mertz et al. (23Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (102) Google Scholar) describe a 33-kDa protein in transiently transfected cells. In both studies, EGFP-tagged Rem localized to nucleoli (22Indik S. Gunzburg W.H. Salmons B. Rouault F. Virology. 2005; 337: 1-6Crossref PubMed Scopus (85) Google Scholar, 23Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (102) Google Scholar), which depended on the presence of the NLS (23Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (102) Google Scholar). Rem and an experimentally truncated version of Rem identical to the signal sequence were shown to function as nuclear export factors for intron-containing transcripts. This activity requires the NLS within the nuclear export factor and the presence of the 3′ long terminal repeat as cis-regulatory sequence in the transcript, suggesting that the export of env transcripts is Rem-dependent (23Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (102) Google Scholar). Beyond Env and Rem, a 14-kDa nucleolar protein named p14 was detected in MMTV bearing S49 and EL-4 T-cell lymphomas using a monoclonal antibody that recognizes an epitope within the predicted signal sequence. Protein purification, mass spectrometry, and microsequencing revealed a mass of 11 kDa and sequence identity to, at least, the N-terminal 81 residues of the Rem/Env signal sequence, including the NLS. The mass of 11 kDa is fully consistent with the calculated molecular weight of the 98 amino acids, which are predicted for the entire signal sequence (21Hoch-Marchaim H. Weiss A.M. Bar-Sinai A. Fromer M. Adermann K. Hochman J. Virology. 2003; 313: 22-32Crossref PubMed Scopus (16) Google Scholar, 24Hoch-Marchaim H. Hasson T. Rorman E. Cohen S. Hochman J. Virology. 1998; 242: 246-254Crossref PubMed Scopus (13) Google Scholar). More recently, p14 was also detected in mouse mammary carcinoma-derived cell lines, and a polyclonal serum identified its immunogenic determinant in some paraffin-embedded human breast cancer sections (25Bar-Sinai A. Bassa N. Fischette M. Gottesman M.M. Love D.C. Hanover J.A. Hochman J. Cancer Res. 2005; 65: 7223-7230Crossref PubMed Scopus (19) Google Scholar). Thus, Rem as well as p14 were detected in the nucleus. However, Rem contains a signal sequence, which suggests ER targeting and translocation of this molecule. Beyond that, the biogenesis of p14 is not known. Therefore, we wanted to investigate how Rem and p14 are generated, if they are processed, and how their localization within the cell is achieved. We have analyzed HeLa cells transiently expressing Rem and found the C-terminal portion of Rem as a glycosylated protein (gp32Rem), whereas the cleaved signal peptide of Rem, here named SPRem, accumulated in nucleoli. Furthermore, we show in a cell-free in vitro system that SPRem is generated at and released from microsomal membranes independent of a processing by the intramembrane protease SPP. Plasmids—With the exception of pEGFP-N1 (Clontech), the plasmids used in this study contain the pRK5rs backbone, which has a cytomegalovirus and an SP6 promoter (26Eaton D.L. Wood W.I. Eaton D. Hass P.E. Hollingshead P. Wion K. Mather J. Lawn R.M. Vehar G.A. Gorman C. Biochemistry. 1986; 25: 8343-8347Crossref PubMed Scopus (205) Google Scholar). An Env encoding plasmid was obtained by re-cloning a PCR amplification product from a full-size MMTV clone (GR strain, kindly provided by E. Buetti, University of Lausanne). pRK5rs-Env encodes MMTV Env amino acids 1–688 without the 5′- and 3′-untranslated regions. Rem cDNA was obtained by reverse transcription of total RNA prepared with the RNeasy kit (Qiagen) from HeLa cells transiently transfected with pRK5rs-Env. The cDNA was amplified using the primers 5′ GGA ATT CAA GAG GAT GCC GAA TCA CCA ATC TGG GTC C and 5′ CGT AAG CTT CCC CTA AGT G, and a fragment of about 900 bp was cloned and sequenced. For pRK5rs-Rem an EcoRI/BglII restriction fragment was cloned into an EcoRI/BglII-linearized pRK5rs-Env thereby replacing the N terminus. pRK5rs-Rem-myc was obtained from pRK5rs-Rem using the primers 5′-CCA TTA TAA GCT GCA ATA AAC and 5′-CCC TCG AGG GCT AAA GAT CTT CTT CAG AAA TAA GTT TCT GTT CAG TGT AGG ACA CTC TCG, including the Myc tag. pRK5rs-SPRem-EGFP-KDEL was obtained by overlap PCR using pRK5rs-Env amplified with 5′ GGA ATT CAA GAG GAT GCC GAA TCA CCA ATC TGG GTC C and 5′ GGT GGC GAC CGG ATA ACT TTC CCC GGT CAC, and pEGFP-N1 (Clontech) amplified with 5′ AGT TAT CCG GTC GCC ACC ATG GTG AGC AAG and 5′ TTA CAG TTC ATC CTT CTT GTA CAG CTC GTC CAT G. pRK5rs-SPRem was cloned from a PCR product using the primers 5′-GGA ATT CAA GAG GAT GCC GAA TCA CCA ATC TGG GTC C and 5′-TTA CCC GGT CAC AGG CGG GGG GCC GAG G. pRK5rs-Rem-T97P, G98W named Remmut was obtained by site-directed mutagenesis with the primers 5′ CCG CCTGTG CCC TGG GAA AGT TAT TGG GCT TAC C and 5′ GGT AAG CCC AAT AAC TTT CCC AGG GCA CAG GCG G. The PCR product was treated with DpnI and used for bacterial transformation. pRK5rs-Prl was cloned using a sub-fragment derived from pGem4-Prl (11Lyko F. Martoglio B. Jungnickel B. Rapoport T.A. Dobberstein B. J. Biol. Chem. 1995; 270: 19873-19878Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). All plasmid inserts were confirmed by full-length sequencing (MWG Biotech) using standard primers. Antibodies—Monoclonal antibody M66 recognizes an epitope between amino acids 25 and 56 in the signal sequence of MMTV Env (see Ref. 24Hoch-Marchaim H. Hasson T. Rorman E. Cohen S. Hochman J. Virology. 1998; 242: 246-254Crossref PubMed Scopus (13) Google Scholar and references therein). B23 antibody (sc-6013), GFP antibody (sc-8334), and lamin A/C antibody (sc-7292) were obtained from Santa Cruz Biotechnology. Myc antibodies were prepared from 9E10 hybridoma cell supernatant using standard procedures. The Sec61β serum is directed against the 9 N-terminal residues of the human protein and was prepared as described previously (27Gorlich D. Rapoport T.A. Cell. 1993; 75: 615-630Abstract Full Text PDF PubMed Scopus (528) Google Scholar). GAPDH antibody (C10) was obtained from Cell Signaling. Sheep α-mouse antibodies conjugated with peroxidase and donkey α-mouse antibodies conjugated with Texas Red were from Dianova. Alexa Fluor® 488 goat α-mouse-, goat α-rabbit-, or donkey α-goat antibodies, Alexa Fluor® 568 goat α-mouse antibodies, and Alexa Flu- or® 546 goat α-rabbit antibodies were from Molecular Probes. Cells and Transient Transfection—HeLa cells (ATCC, CCL-2) were grown in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 10% fetal calf serum, and 2 mm l-glutamine. Cells were controlled for the absence of mycoplasma using standard PCR. 1 × 104 cells were seeded in 12-well slots on glass coverslips for immunofluorescence, and 2 × 105 cells were seeded in 6-well slots for Western blot analyses or metabolic labeling. 18 h after calcium phosphate transfection (28Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar), cells were supplied with fresh medium. Immunofluorescence—48 h post-transfection, HeLa cells were fixed for 5 min with 2% formaldehyde in PBS supplemented with 125 mm sucrose and subsequently for 20 min in ice-cold methanol at -20 °C. Unreacted groups were protected by treatment with 0.1 m glycine in PBS. Blocking and incubations with antibodies were done for 1 h at room temperature with 10% fetal calf serum in PBS and with 5% fetal calf serum, respectively. Coverslips were mounted with Mowiol containing 0.1 μg/ml 4′,6-diamidino-2-phenylindole. Confocal microscopy was done with a Leica TCS SP2, using a 63× HCX PL APO oil immersion objective (numerical aperture 1.4). Excitation laser lines were 488 nm (argon laser) and 561 nm (diode pumped solid state laser). All images are single plane images produced with the Leica confocal software. Cell Lysis and Western Blot Analyses—42 h post-transfection cells were lysed in SDS sample buffer (50 mm Tris/HCl, pH 6.8; 12% glycerol; 4% SDS; 0.01% Serva blue G and 100 mm dithiothreitol) and separated on a 16.5% Tris/Tricine SDS-PAGE (T, 49.5%; C, 3%) (29Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10478) Google Scholar). For Western blots, blocking and antibody incubation were done with 0.25% gelatin in 1× TBS-T (50 mm Tris/HCl, pH 7.5; 150 mm NaCl; 5 mm EDTA, pH 8.0; 0.05% Triton X-100). Immunodetection was done with BM chemiluminescence blotting substrate (POD, Roche Applied Science). Metabolic Labeling and Immunoprecipitation—42 h post-transfection, cells were depleted of methionine and cysteine for 2 h (Dulbecco's modified Eagle's medium without l-methionine and l-cysteine, containing 4.5 g/liter glucose, 2 mm l-glutamine, and 10% fetal calf serum dialyzed against PBS to remove small molecules up to 12 kDa). Labeling was done with 75 μCi/ml Redivue Pro-mix l-35S cell labeling mix (GE Healthcare) for various periods of time as detailed in the figure legends. For the analysis of the glycosylation pattern, cells were lysed in SDS-containing lysis buffer (50 mm Tris/HCl, pH 7.5; 500 mm NaCl; 1% (w/v) sodium deoxycholate; 1% (v/v) Nonidet P-40; 0.1% (w/v) SDS; 2 mm EDTA; 1 mm phenylmethylsulfonyl fluoride; 10 μg/ml leupeptin; 10 μg/ml chymostatin; 10 μg/ml pepstatin). The lysate was spun through a QIAshredder column (Qiagen, Hilden, Germany) according to the protocol of the manufacturer and, additionally, cleared by centrifugation at 16,000 × g for 20 min at 4 °C. For immunoprecipitation, the lysates were diluted with IP buffer (20 mm Hepes, pH 7.5; 500 mm NaCl; 10% (v/v) glycerol; 0.1% (v/v) Triton X-100), protein A-Sepharose beads (Amersham Biosciences) and antibodies were added. Samples were rotated for 3 h, and the beads were washed four times with IP buffer. Endoglycosidase H treatment was done as suggested by the manufacturer (New England Biolabs). For fractionation, cells were first permeabilized for 5 min with 0.02% digitonin (50 mm Hepes, pH 7.5; 150 mm NaCl; 1.5 mm MgCl2; 10% (v/v) glycerol; 0.02% (w/v) digitonin; 1 mm EGTA; 1 mm phenylmethylsulfonyl fluoride; 10 μg/ml leupeptin; 10 μg/ml chymostatin; 10 μg/ml pepstatin) to release cytosolic proteins. The permeabilized cells were pelleted (5 min, 16,000 × g, 4 °C) and lysed with 1% Triton X-100 (50 mm Hepes, pH 7.5; 150 mm NaCl; 1.5 mm MgCl2; 10% (v/v) glycerol; 1% (v/v) Triton X-100; 1 mm EGTA; 1 mm phenylmethylsulfonyl fluoride; 10 μg/ml leupeptin; 10 μg/ml chymostatin; 10 μg/ml pepstatin) to solubilize membrane proteins. Nonsoluble material was pelleted as detailed above, and the pellet was resuspended in SDS-containing lysis buffer. This lysate was cleared by centrifugation (20 min, 16.000 × g, 4 °C). Following immunoprecipitation, proteins were separated on a Tris/Tricine SDS-PAGE as described above. Dried gels were analyzed by PhosphorImaging (Bio-Rad PMI system for Fig. 2B, Fuji BAS 1500 for every other autoradiogram). To control the fractionation, nontransfected cells or cells expressing EGFP were lysed as described, and lysates were analyzed by Western blotting. Densitometric and Statistical Analyses—Densitometric analysis was done with ImageJ. Each signal, represented by a defined area, was corrected for background by subtracting a signal from an identical area within the same lane. For statistical analyses, relative amounts were calculated, i.e. signals from each time point were adjusted to 100%. Relative amounts are given in percent with standard deviation. Statistical analysis was done with JMP (SAS Institute). Data were analyzed by one-way analysis of variance and Student's t tests for unpaired groups. p values are given in the legend. In Vitro Translation/Translocation Assay—Rem encoding plasmid DNA was linearized, purified, and used for in vitro transcription with SP6 polymerase as described before (11Lyko F. Martoglio B. Jungnickel B. Rapoport T.A. Dobberstein B. J. Biol. Chem. 1995; 270: 19873-19878Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Transcripts were treated with DNase (Promega) and purified using G-25 columns (GE Healthcare). In vitro translation was done for 30 min at 30 °C in 10-μl reactions using rabbit reticulocyte lysate (Promega), Redivue Pro-mix l-35S cell labeling mix (GE Healthcare), and 1–1.5 eq canine pancreas rough microsomes produced according to the protocol from Walter and Blobel (30Walter P. Blobel G. Methods Enzymol. 1983; 96: 84-93Crossref PubMed Scopus (478) Google Scholar). Signal peptide peptidase inhibitor (Z-LL)2-ketone (Calbiochem) was dissolved in Me2SO and added as indicated. In vitro reactions were precipitated with ammonium sulfate by adding 2 volumes of saturated ammonium sulfate solution and precipitation for 20 min on ice. The precipitate was pelleted by centrifugation at 16,000 × g for 5 min and resuspended in distilled H2O. Proteins were again precipitated with 2 volumes of ice-cold absolute ethanol, pelleted, and resuspended in SDS sample buffer (50 m Tris/HCl, pH 6.8; 10 mm EDTA; 5% glycerol; 2% SDS; 0.01% bromphenol blue). To separate the microsomes from the supernatant, in vitro reaction samples were layered on top of a 50-μl cushion (50 mm Hepes-KOH, pH 7.6; 750 mm KOAc; 10 mm Mg(OAc)2; 1 mm dithiothreitol; 500 mm sucrose), and membranes were pelleted by 5 min of centrifugation at 100,000 × g and at 4 °C (Beckman TLA 100 rotor or Sorvall S100-AT3 rotor). For SDS-gel electrophoresis, the supernatant was precipitated with ammonium sulfate as described above, and the pellet was directly resuspended in SDS sample buffer. The samples were separated in 10–17% gradient gels (T, 30%; C, 2.6%) (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207192) Google Scholar). For immunoprecipitation, the supernatant was diluted with lysis buffer containing 1% Triton X-100, whereas the pellet was directly resuspended in this lysis buffer. Immunoprecipitation was done as described above. For the characterization of the release, in vitro translation was carried out as described above but stopped after 15 min by the addition of 1.25 mm cycloheximide (Sigma). Signal peptide release was analyzed for various periods of time as well as under different temperature conditions (either on ice or at 30 °C). The samples were analyzed by immunoprecipitation, SDS-PAGE, and autoradiography. Cellular Localization of the Rem N- and C-terminal Portions—In previous studies, a Rem-EGFP fusion protein was used for localization, and a nucleolar EGFP staining was detected (22Indik S. Gunzburg W.H. Salmons B. Rouault F. Virology. 2005; 337: 1-6Crossref PubMed Scopus (85) Google Scholar, 23Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (102) Google Scholar). We wanted to determine the localization of both the N- and C-terminal portions of Rem by immunofluorescence microscopy. To this end, we transiently expressed C-terminally Myc-tagged Rem (Rem-myc) in HeLa cells and stained with the monoclonal antibody M66, which recognizes an epitope in the predicted signal sequence (21Hoch-Marchaim H. Weiss A.M. Bar-Sinai A. Fromer M. Adermann K. Hochman J. Virology. 2003; 313: 22-32Crossref PubMed Scopus (16) Google Scholar, 24Hoch-Marchaim H. Hasson T. Rorman E. Cohen S. Hochman J. Virology. 1998; 242: 246-254Crossref PubMed Scopus (13) Google Scholar), that is the N-terminal portion, as well as an antibody recognizing the C-terminal Myc tag (Fig. 1B). The M66 antibody detected its epitope mainly in the nucleus, where a major fraction co-localized with the nucleolar marker B23 (Fig. 1B, upper panel). The C-terminal Myc tag of Rem-myc was detected in a reticular structure, which was also stained by an antibody against Sec61β, an ER marker. Additionally, nucleolar staining was obtained with the anti-Myc antibody (Fig. 1B, bottom panel). Thus, the localization of the N- and C-terminal portions of Rem-myc in nucleoli indicates that full-length Rem-myc is imported into the nucleus. However, Rem-myc is not exclusively a nuclear protein, because the C-terminal portion can also be found at the ER. Next, we analyzed whether the predicted signal sequence of Rem/Env is able to direct an unrelated, otherwise cytosolic protein to the ER. To this end, we fused the N-terminal 101 amino acids of Rem/Env (including the 98-amino acid-long predicted signal sequence) to EGFP with a C-terminal KDEL motif. The KDEL motif retains translocated EGFP in the ER, where it can be detected. HeLa cells were transiently transfected with this construct named SPRem-EGFP-KDEL. By immunofluorescence microscopy, EGFP staining was observed primarily in a reticular structure typical for the ER, but it was also found in nucleoli (Fig. 1C). The M66 epitope was again detected in the nucleus, mainly in nucleoli. Thus, the Rem/Env-derived signal sequence can target EGFP-KDEL to the ER. As a consequence of targeting, the signal sequence may be cleaved, and nucleolar M66 staining may in part be due to the presence of cleaved signal sequences. The EGFP staining found in nucleoli along with the nucleolar M66 staining again suggests that a certain amount of the precursor molecules was transported to the nucleus. Biochemical Identification of Rem and Its Derivatives—Next, we wanted to biochemically identify the antigens detected by the M66, anti-Myc, and anti-GFP antibody. We transfected HeLa cells with Rem-myc or SPRem-EGFP-KDEL and analyzed cell lysates by immunoblotting. In both cases, the M66 antibody detected a 14-kDa protein, termed p14 (Fig. 2A, p14, lanes 2 and 4). The signal sequence is the only common sequence of the proteins expressed from these two constructs, which strongly suggests that p14 is the cleaved signal sequence of Rem. Besides p14, the M66 antibody detected a 42-kDa protein in cells expressing SPRem-EGFP-KDEL (Fig. 2A, lane 4) and a low amount of a 38-kDa protein in cells expressing Rem-myc, respectively (lane 2). To identify the C-terminal portion, Rem-myc expressing cells were analyzed with the anti-Myc antibody. We detected a 38- and a 33-kDa protein (Rem-myc and p33Rem-myc, Fig. 2A, lane 7). Thus, the 38-kDa protein detected by both antibodies represents the precursor Rem-myc. Because the 33-kDa protein was not detected by the M66 antibody, it does not contain the signal sequence and represents the signal sequence-cleaved protein p33Rem-myc. Similarly, in cells expressing SPRem-EGFP-KDEL, the anti-GFP antibody detected a signal sequence-cleaved EGFP-KDEL protein (28 kDa) as well as the precursor protein (SPRem-EGFP-KDEL, 42 kDa), which was also detected with the M66 antibody (lane 9). In summary, precursor molecules as well as mature proteins along with cleaved signal sequences can be detected in cells overexpressing Rem-myc or SPRem-EGFP-KDEL. Consistent with ER targeting and signal sequence cleavage, the C-terminal portion of Rem-myc (p33Rem-myc) is most likely translocated across the ER membrane. Because Rem contains two putative N-glycosylation sites (compare Fig. 1A), we investigated whether p33Rem-myc is a glycoprotein. To this end, we expressed Rem-myc in HeLa cells, metabolically labeled the cells, and analyzed lysates by immunoprecipitation and treatment with endoglycosidase H, which removes N-linked sugar moieties. A 38-kDa protein representing Rem-myc was detected independent of endoglycosidase H treatment. The 33-kDa protein detected in Rem-myc expressing cells was reduced to 27 kDa (p27Rem-myc) by treatment with endoglycosidase H (Fig. 2B). This indicates two glycosylation events, which alter the molecular mass by about 2–3 kDa each. In an independent experiment, we found a similar reduction in molecular mass by 5–6 kDa when analyzing cells treated with tunicamycin, an inhibitor of N-glycosylation (data not shown). Thus, p33Rem-myc (in Fig. 2A) is a glycoprotein, which confirms its translocation into the ER lumen. We named this protein gp33Rem-myc in contrast to the precursor that was previously named Rem or, in our case, Rem-myc. Next, we wanted to determine whether the cleave" @default.
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• W2038049042 title "The Signal Peptide of the Mouse Mammary Tumor Virus Rem Protein Is Released from the Endoplasmic Reticulum Membrane and Accumulates in Nucleoli" @default.
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