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• W2048423346 abstract "In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions. In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions. endoplasmic reticulum signal recognition particle ribosome-nascent polypeptide complex rough microsomes post-ribosomal supernatant preprolactin Renilla luciferase green fluorescent protein 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid dithiothreitol polyacrylamide gel electrophoresis nascent polypeptide-associated complex In mammalian cells, cytoplasmic ribosomes engaged in the synthesis of secretory or membrane protein precursors are selected for targeting to the endoplasmic reticulum (ER)1 membrane. While free in the cytoplasm, such ribosome-nascent polypeptide complexes (RNCs) are recognized by the signal recognition particle (SRP) and targeted to the ER membrane via interaction with the SRP receptor (1Meyer D.I. Krause E. Dobberstein B. Nature. 1982; 297: 647-650Crossref PubMed Scopus (323) Google Scholar, 2Gilmore R. Walter P. Blobel G. J. Cell Biol. 1982; 95: 470-477Crossref PubMed Scopus (300) Google Scholar, 3Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (719) Google Scholar). The targeting reaction culminates in the association of RNCs with the translocon, the cohort of ER membrane components that mediates protein translocation, and transfer of the nascent polypeptide across the ER membrane ensues (3Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (719) Google Scholar, 4Matlack K.E.S. Mothes W. Rapoport T.A. Cell. 1998; 92: 381-390Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 5Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Crossref PubMed Scopus (501) Google Scholar). For translocation to proceed, the signal sequence must engage components of the ER membrane, fulfilling a postulated second signal sequence recognition event (6Jungnickel B. Rapoport T.A. Cell. 1995; 82: 261-270Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 7Belin D. Bost S. Vassalli J.-D. Strub K. EMBO J. 1996; 15: 468-478Crossref PubMed Scopus (84) Google Scholar, 8Zheng T. Nicchitta C.V. J. Biol. Chem. 1999; 274: 36623-36630Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Via the targeting phase of the translocation cycle, SRP and biosynthetically active ribosomes associate with the ER membrane. How are these components recycled back to the cytoplasm to complete an exchange cycle? It is now established that SRP release from the ER membrane occurs through the combined GTP binding and GTPase activities of SRP and the SRP receptor complex (9Rapiejko P.J. Gilmore R. Cell. 1997; 89: 703-713Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Stroud R.M. Walter P. Curr. Opin. Struct. Biol. 1999; 9: 754-759Crossref PubMed Scopus (79) Google Scholar). In contrast, little is known regarding the fate of the membrane-bound ribosome following the termination of protein translation. Many models depict that upon termination, the ribosome dissociates into its component 60 S and 40 S subunits and subsequently detaches from the ER membrane (11Mechler B. Vassalli P. J. Cell Biol. 1975; 67: 25-37Crossref PubMed Scopus (28) Google Scholar, 12Blobel G. Dobberstein B. J. Cell Biol. 1975; 67: 835-851Crossref PubMed Scopus (1838) Google Scholar). In these models, the release of ribosomal subunits from the ER membrane supports a ribosome cycle in which ribosomes engaged in the synthesis of signal sequence-bearing nascent polypeptides are selected from a free cytoplasmic pool, trafficked to the ER membrane, and subsequent to protein translocation, recycled back to the cytoplasm. This series of reactions provides a rationale for the segregation of ribosomes into free and membrane-bound pools. Although ribosome release from the ER membrane represents a fundamental element of the protein translocation cycle, it remains to be determined experimentally how ribosome release from the ER membrane is coupled to the termination of protein translation. In vitro studies with rat liver rough microsomes (RM) have demonstrated that following treatment with puromycin, free 40 S ribosomal subunits are capable of exchanging with membrane-bound 40 S subunits, whereas free 60 S subunits do not participate in exchange reactions with membrane-bound 60 S subunits (13Borgese D. Blobel G. Sabatini D.D. J. Mol. Biol. 1973; 74: 415-438Crossref PubMed Scopus (41) Google Scholar). These data suggest that following puromycin-induced termination, the 60 S subunits remain in stable association with the ER membrane in the absence of protein synthesis. Indeed, in tissue culture cells it has been proposed that the release of 60 S ribosomal subunits from the ER membrane is kinetically delayed from the termination of protein synthesis (14Mechler B. Vassalli P. J. Cell Biol. 1975; 67: 16-37Crossref PubMed Scopus (14) Google Scholar, 48Seiser R.M. Nicchitta C.V. J. Biol. Chem. 2000; 275: 33820-333827Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Furthermore, experiments in which RM are solubilized with digitonin and subsequently treated with puromycin indicate that membrane-bound ribosomes remain associated with the translocon (15Görlich D. Prehn S. Hartmann E. Kalies K.-U. Rapoport T.A. Cell. 1992; 71: 489-503Abstract Full Text PDF PubMed Scopus (366) Google Scholar). These data suggest that ribosome release from the ER membrane may be subject to as yet undefined regulatory influences and further emphasize the need for experimental analysis of the regulation of ribosome dissociation from the ER membrane. Our studies utilized the well established in vitrotranslation/translocation system consisting of rabbit reticulocyte lysate and canine pancreas rough microsomes. In examining the distribution of ribosomes during the elongation and termination stages of protein synthesis, we report that ribosome release from the ER membrane does not accompany termination. Rather, ribosome release was observed to occur in response to the synthesis of a cytosolic protein on a membrane-bound ribosome. In addition, we report that secretory precursors whose synthesis is initiated on membrane-bound ribosomes can access the translocon in an SRP receptor-independent manner. Canine pancreas RM were isolated and treated withStaphylococcus aureus nuclease (Calbiochem) as described (16Walter P. Blobel G. Methods Enzymol. 1983; 96: 84-93Crossref PubMed Scopus (480) Google Scholar). Nuclease treatment was required to degrade microsome-associated mRNAs, thereby increasing the translation of exogenously added mRNA by membrane-bound ribosomes. However, this treatment decreased the overall translational capacity of the RM, presumably by stranding ribosomes on cleaved mRNA fragments, as reported previously for free reticulocyte lysate ribosomes (17Jackson R.J. Hunt T. Methods Enzymol. 1983; 96: 50-73Crossref PubMed Scopus (397) Google Scholar). To remove any contaminating free ribosomes from RM preparations, 100 equivalents (eq; as defined in Ref. 16Walter P. Blobel G. Methods Enzymol. 1983; 96: 84-93Crossref PubMed Scopus (480) Google Scholar) of RM were diluted 5-fold with 2.5 m sucrose, 25 mm K-HEPES (pH 7.2), 350 mm KOAc, 5 mm Mg(OAc)2, and 1 mm DTT. This solution is then overlaid with 750 μl of 1.9m sucrose, 25 mm K-HEPES (pH 7.2), 350 mm KOAc, 5 mm Mg(OAc)2, 1 mm DTT, and 200 μl of the same buffer lacking sucrose. The sample was centrifuged in an SW55 rotor (Beckman Instruments, Palo Alto, CA) at 55,000 rpm for 4 h at 4 °C. Under these conditions, the RM float, whereas any contaminating free ribosomes sediment to yield a pellet fraction. The upper 400 μl of the gradient were collected and diluted 3-fold with RM buffer (250 mmsucrose, 25 mm K-HEPES (pH 7.2), 50 mm KOAc, 5 mm Mg(OAc)2, and 1 mm DTT) and were centrifuged at 60,000 rpm for 10 min at 4 °C in a TLA100.2 rotor (Beckman Instruments). The membrane pellet was then resuspended in RM buffer. For some experiments, SRP receptor was inactivated by mild proteolysis prior to flotation of the RM. 100 eq of RM were diluted 20-fold with RM buffer, and chymotrypsin (Worthington) was then added to a final concentration of 5 μg/ml. The sample was placed on ice for 30 min; the digestion was subsequently quenched by addition of phenylmethylsulfonyl fluoride to 1 mm, and membranes were pelleted by centrifugation at 60,000 rpm for 10 min at 4 °C in a TLA100.2 rotor. Pellets were resuspended in 100 μl of RM buffer and processed for flotation as described above. Ribosomes were removed from reticulocyte lysate (Promega, Madison, WI) by centrifugation at 80,000 rpm for 30 min at 4 °C in a TLA100 rotor (Beckman Instruments). The supernatant was removed and then centrifuged again under identical conditions to ensure that ribosomes and ribosomal subunits were quantitatively depleted. The resulting ribosome-free post-ribosomal supernatant (PRS) was aliquoted and frozen in liquid nitrogen, and the pelleted ribosomes were resuspended in RM buffer. To fuse N-terminal luciferase extensions with pPL, the pPL-encoding plasmid pGEMBP1 (18Connolly T. Gilmore R. J. Cell Biol. 1986; 103: 2253-2261Crossref PubMed Scopus (115) Google Scholar) was first modified using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). An NcoI site was created at the extreme N terminus of the pPL coding region, using the primers 5′-GGC-AGA-CTC-TAG-ACC-ATG-GAC-AGC-AAA-GGT-TC-3′ (sense primer) and 5′-GAA-CCT-TTG-CTG-TCC-ATG-GTC-TAG-AGT-CTG-CC-3′ (antisense primer). An internal NcoI site was removed using the primers 5′-GGC-AAA-GGG-TTC-ATT-ACA-ATG-GCC-CTC-AAC-AGC-3′ (sense primer) and 5′-GCT-GTT-GAG-GGC-CAT-TGT-AAT-GAA-CCC-TTT-GCC-3′ (antisense primer). Neither modification altered the pPL amino acid sequence. Polymerase chain reaction products encoding the N-terminal 39, 59, or 93 amino acids of Rluc and flanked with NcoI sites were produced using the luciferase-containing plasmid pRL-null (Promega, Madison, WI) and the primers 5′-GTG-ACT-CCA-TGG-ATT-CGA-AAG-TTT-ATG-ATC-CAG-3′ (sense primer) and either 5′-GAG-ACA-CCA-TGG-TTT-CTG-AAT-CAT-AAT-AAT-TAA-TAA-ATG-3′ (antisense primer for Rluc40), 5′-GAG-ACA-CCA-TGG-TCC-ATA-AAT-AAG-AAG-AGG-CCG-3′ (antisense primer for Rluc60), or 5′-GAG-ACA-CCA-TGG-TAA-GTA-ACC-TAT-AAG-AAC-C-3′ (antisense primer for Rluc94). Rluc polymerase chain reaction fragments and the modified pGEMBP1 vector were digested with NcoI and ligated together. Ligation products were transformed into the Escherichia colistrain DH5α, and positive clones were isolated. A threonine residue was encoded between the Rluc and pPL coding regions as a result of the cloning strategy. Transcripts coding for pPL86 were generated by linearizing pGEMBP1 with PvuII. For pPL55, the plasmid was linearized with FokI. Transcripts coding for Rluc94 were generated by linearizing pRL-null withBclI. For Rluc56, the plasmid was linearized usingEarI, and for Rluc33, the plasmid was linearized withVspI. GFP146 was created by linearizing the GFP-containing plasmid (provided by T. Meyer) with XhoI. All restriction enzymes were obtained from either New England Biolabs (Beverly, MA) or Promega. Transcription was carried out using a MEGAscript T7 kit (Ambion, Austin, TX). Translations were carried out as described (19Nicchitta C.V. Blobel G. J. Cell Biol. 1989; 108: 789-795Crossref PubMed Scopus (53) Google Scholar). Where indicated, ribosome-free reticulocyte lysate was substituted for unfractionated lysate, and RM were used as the source of ribosomes. Translations were conducted at 25 °C for 30 min using35S-labeled Pro-Mix (Amersham Pharmacia Biotech). Completed translation mixtures (20 μl) were diluted to 100 μl with 110 mmKOAc, 25 mm K-HEPES (pH 7.2), and 2.5 mmMg(OAc)2 and were then layered upon a 100-μl 0.5m sucrose cushion in the same buffer. Membranes were pelleted by centrifugation at 40,000 rpm for 5 min at 4 °C in a TLA100 rotor and were resuspended directly into SDS-PAGE sample buffer (0.5 m Tris, 5% SDS, 100 mmβ-mercaptoethanol). The supernatants, containing unbound ribosomes, were precipitated by addition of saturated ammonium sulfate to a final concentration of 66% and were washed once with 10% trichloroacetic acid before being resuspended in sample buffer. Samples were then processed for SDS-PAGE. Gels were visualized using a Fujix MacBAS1000 PhosphorImager, and digital images were prepared using Adobe Photoshop 4.0. All quantitation of the PhosphorImager output was performed using Fujix MacBAS version 2.0 software. Alternatively, RNC association with membranes was determined by flotation centrifugation. Completed translation mixtures (40 μl) containing free ribosomes were placed on ice, and cycloheximide was added to 1 mm to ensure that further translation could not occur. 4 eq of carrier RM were then added, and samples were incubated 10 min on ice, followed by 20 min at 25 °C. Completed translation mixtures using RM-bound ribosomes as the translation source were treated similarly, except additional RM were not added following the 30-min translation. Samples were then diluted to 100 μl with RM buffer and were mixed with 400 μl of 2.5 m sucrose, 150 mm KOAc, 25 mm K-HEPES (pH 7.2), 2.5 mm Mg(OAc)2, and 1 mm DTT. Samples were overlaid with 1 ml of 1.9 m sucrose and 200 μl of 250 mm sucrose, both containing 150 mm KOAc, 25 mm K-HEPES (pH 7.2), 2.5 mmMg(OAc)2, and 1 mm DTT, and were centrifuged in an SW55 rotor at 55,000 rpm for 4 h at 4 °C. For some samples, KOAc concentrations were adjusted to 0.5 m and were incubated for 15 min on ice immediately prior to flotation centrifugation. Sucrose solutions containing 0.5 m KOAc were then used for flotation centrifugation of these samples. Following centrifugation, all samples were frozen in liquid nitrogen, cut into thirds using a sharp knife, and prepared for SDS-PAGE as described above. Release of polypeptides from ribosomes was conducted by diluting completed translation mixtures (20 μl) to 50 μl with 110 mm KOAc, 25 mm K-HEPES (pH 7.2), and 2.5 mm Mg(OAc)2 and then adding puromycin to 1 mm. Samples were incubated on ice for 5 min and then at 25 °C for 15 min. To analyze polypeptide accessibility to exogenous protease, proteinase K was added to translation mixtures to a final concentration of 100 μg/ml, and samples were incubated on ice for 30 min. Where indicated, CHAPS was included at a final concentration of 0.5%. Immunoprecipitation of pPL nascent polypeptides using anti-pPL polyclonal antisera (U. S. Biochemical Corp.) was conducted as described (20Anderson D.J. Blobel G. Methods Enzymol. 1983; 96: 111-120Crossref PubMed Scopus (208) Google Scholar). Samples were layered upon 10-ml 10–30% sucrose gradients containing 25 mm K-HEPES (pH 7.2), 150 mm KOAc, 5 mm Mg(OAc)2, 1 mm DTT, and 1 mm cycloheximide. Samples were centrifuged at 40,000 rpm for 2.5 h at 4 °C in an SW41 rotor (Beckman Instruments). Gradients were manually fractionated by puncturing the tube bottoms, and fractions were trichloroacetic acid-precipitated and analyzed by SDS-PAGE. As noted in the Introduction, little is known regarding the mechanism by which ribosomes and/or ribosomal subunits dissociate from the ER membrane following the termination of protein synthesis. The central role of SRP in regulating the association of biosynthetically active ribosomes with the ER membrane is, however, well established. To investigate the mechanism of ribosome dissociation from the ER membrane, we utilized an experimental system that in prior studies was used to establish the mechanism of SRP-dependent ribosome-membrane association (21Walter P. Blobel G. J. Cell Biol. 1981; 91: 551-556Crossref PubMed Scopus (259) Google Scholar). Necessarily, it was essential to these studies that the analysis be strictly limited to membrane-bound ribosomes, and so the canine pancreas microsome fraction used in these studies was further purified by flotation centrifugation through a high salt buffer. This purification step removes unbound and inactive bound ribosomes, leaving only those ribosomes that are tethered to the membrane by the presence of a nascent polypeptide (22Adelman M.R. Sabatini D.D. Blobel G. J. Cell Biol. 1973; 56: 206-229Crossref PubMed Scopus (213) Google Scholar). To constrain further the analysis to membrane-bound ribosomes, a ribosome-free supernatant fraction of reticulocyte lysate was utilized as a source of the initiation, elongation, and termination factors necessary to support the protein translation cycle. As shown in Fig.1, when the protein translation activity of membrane-bound ribosomes was assayed in the presence of radiolabeled methionine, a number of prominent microsome-specific translation products were produced, and these nascent polypeptides were translocated into the ER lumen, as indicated by resistance to degradation by proteinase K in the absence, but not the presence, of detergent (Fig. 1 A). As the data in Fig. 1 A indicate, membrane-bound ribosomes retain protein translation activity. Given the presence of active, stably bound ribosomes on the membrane fraction, it was possible to investigate experimentally the process by which the membrane detachment of such ribosomes was regulated. To do so, the ability of the membrane-bound ribosomes to initiate translation of exogenously added mRNA was evaluated. As a control experiment, the addition of mRNA encoding truncated forms of preprolactin (pPL86; 86 amino acids), Renilla luciferase (Rluc94; 94 amino acids), or green fluorescent protein (GFP146; 146 amino acids) to the ribosome-free reticulocyte lysate PRS, in the absence of RM, did not yield protein translation (Fig. 1 B, lanes 1 and5). In contrast, when salt-washed, purified RM were present, translation of pPL86, Rluc94, and GFP146 was observed (Fig. 1 B, lane 2). The faint band apparent in all lanes represents the non-enzymatic labeling of globin and was observed in the presence or absence of cycloheximide (data not shown). To more rigorously determine whether the observed protein translation activity was derived from the membrane-bound ribosomes, the purified RM were pelleted using centrifugation conditions that retain free ribosomes and/or ribosomal subunits in the supernatant and allow recovery of the RM in the pellet fraction. The pellet and supernatant fractions from such centrifugations were then separately assayed for translation activity. As shown in Fig. 1 B, lanes 3 and 4, the translation of exogenous mRNA was wholly initiated by membrane-bound ribosomes (Fig. 1 B, lanes 3 and4). The observation that membrane-bound ribosomes could initiate new rounds of translation was consistent with either of two models. 1) In the presence of PRS, membrane-bound ribosomes undergo run-off protein translation, yielding the release of the membrane-bound ribosomal subunits into a free ribosomal subunit pool and the subsequent initiation of protein synthesis on free ribosomes. 2) The ribosomes/ribosomal subunits remain in association with the ER membrane following termination, with initiation occurring on the bound ribosomes. To differentiate between these two possibilities, RM were preincubated under run-off translation conditions to allow the bound ribosomes to undergo termination reactions and were subsequently fractionated, as described above. As shown in Fig. 1 B, lanes 7 and 8, translation activity was again solely recovered in the membrane-bound ribosomal population, indicating that run-off translation does not result in the detachment of translationally active ribosomes from the ER membrane. These results were further corroborated by immunoblots for ribosomal proteins L3/L4, wherein we were unable to identify the release of ribosomes into the supernatant fraction (data not shown). Significantly, the data presented in Fig. 1 indicate that membrane-bound ribosomes can initiate translation of mRNA regardless of whether the encoded protein possesses (pPL86) or lacks (Rluc94 and GFP146) an ER signal sequence. The experiments depicted in Fig. 1 indicate that ribosomes engaged in the synthesis of endogenous mRNAs did not dissociate from the ER membrane following run-off translation. That such ribosomes were clearly capable of de novo protein synthesis suggests, but does not prove, that run-off translation had occurred. Because of the difficulties in unequivocally ascertaining the translation status of the endogenous mRNAs, we focused our analysis on those ribosomes engaged in the de novo synthesis of proteins from exogenous mRNAs. To determine the fates of ribosome-nascent polypeptide complexes following thede novo initiation of protein synthesis on membrane-bound ribosomes, a series of fractionation experiments was performed. In control experiments, proteins were synthesized by free reticulocyte lysate ribosomes in the presence or absence of RM and were then subjected to centrifugation to separate membrane-associated from free RNCs (Fig. 2 A, lanes 1–4). When synthesized in the absence of RM, the majority of all RNCs were recovered in the supernatant fraction (Fig. 2 A, lanes 1 and 2). Minor amounts of RNCs were recovered in the pellet fraction in the absence of membranes, as is commonly observed in such experiments (23Siegel V. Walter P. EMBO J. 1988; 7: 1769-1775Crossref PubMed Scopus (76) Google Scholar, 24Murphy III, E.C. Zheng T. Nicchitta C.V. J. Cell Biol. 1997; 136: 1213-1226Crossref PubMed Scopus (22) Google Scholar). When synthesis on free ribosomes was performed in the presence of RM, only RNCs engaged in the synthesis of signal sequence-bearing nascent polypeptides (pPL86) bound to the membranes; RNCs engaged in the synthesis of Rluc94 and GFP146 were recovered in the supernatant fraction (Fig. 2 A, lanes 3 and4). These data are entirely consistent with previous studies in demonstrating that ribosomes engaged in the synthesis of secretory precursors associate with the ER membrane, whereas ribosomes engaged in the synthesis of cytosolic proteins remain in the supernatant (cytosol) fraction. As an additional note, in these and related experiments, protein translation can occur on both free and membrane-bound ribosomes. To determine the relative activities of the two modes of translation, the ribosome content and thus translation activity of the reticulocyte lysate fraction were titrated to define the point of translation equivalence with the membrane-bound ribosome fraction. The results of these experiments indicate that under standard reaction conditions (2 eq of RM (16Walter P. Blobel G. Methods Enzymol. 1983; 96: 84-93Crossref PubMed Scopus (480) Google Scholar) in 20 μl of translation medium) lysate-derived free ribosomes provide 10-fold greater translation activity versus membrane-bound ribosomes (data not shown). Thus, translation occurs predominantly on free ribosomes. Similar experiments were then performed under conditions where membrane-bound ribosomes were the sole source of ribosomes. Again, nascent pPL86 was recovered in association with the membranes; Rluc94 and GFP146 were, however, recovered in the supernatant fraction (Fig. 2 A, lanes 5 and 6). In the experiments described above, Rluc94 and GFP146 were synthesized on artificially truncated mRNAs lacking termination codons and thus should remain predominantly ribosome-associated. To determine whether Rluc94 and GFP146 translation products were released from the membrane in the context of intact RNCs, supernatant fractions, obtained by centrifugation of translation reactions conducted with membrane-bound ribosomes, were centrifuged through 10–30% sucrose gradients, and the distribution of the translation products was determined. An immunoblot for ribosomal proteins L3/L4 indicates the migration of 80 S ribosomes in the gradients (Fig. 2 B, upper panel). In comparing the relative migration of the ribosomal peak and the radiolabeled nascent polypeptides, it is clear that the majority of the Rluc94 and GFP146 nascent polypeptides had been released from the membrane as intact RNCs (Fig. 2 B, middle and lower panels). Thus, translation of proteins lacking a signal sequence on membrane-bound ribosomes yields the detachment of intact RNCs from the ER membrane. Following the translation of pPL86 on membrane-bound ribosomes, completed pPL86 nascent polypeptides sedimented in association with the ER membrane (Fig. 2 A, lanes 5 and 6). However, the mechanism by which membrane association was conferred was not clear. Did the pPL86 RNCs remain membrane-bound throughout the translation period? Alternatively, did the RNCs release from the membrane early in synthesis, only to re-target by the SRP-dependent pathway? To address these questions, the following hypothesis was examined; if membrane-bound ribosomes engaged in the de novo protein synthesis of secretory precursors dissociate from the membrane early in synthesis, such RNCs would require the SRP receptor activity for re-targeting to the membrane. A stringent test of this hypothesis was performed by inhibiting SRP receptor activity. It is well established that the cytoplasmic domain of the SRP receptor can be inactivated by mild proteolysis, under conditions where the remainder of the translocation machinery is intact (25Walter P. Jackson R.C. Marcus M.M. Lingappa V.R. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1795-1799Crossref PubMed Scopus (68) Google Scholar, 26Meyer D.I. Dobberstein B. J. Cell Biol. 1980; 87: 498-502Crossref PubMed Scopus (75) Google Scholar, 27Meyer D.I. Dobberstein B. J. Cell Biol. 1980; 87: 503-508Crossref PubMed Scopus (81) Google Scholar, 28Gilmore R. Blobel G. Walter P. J. Cell Biol. 1982; 95: 463-469Crossref PubMed Scopus (239) Google Scholar, 29Hortsch M. Avossa D. Meyer D.I. J. Cell Biol. 1986; 103: 241-253Crossref PubMed Scopus (61) Google Scholar). Therefore, RM were treated at a low concentration of chymotrypsin (5 μg/ml) to degrade the SRP receptor (SRα), while leaving the translocon component Sec61α intact (Fig. 3 A). From the data presented in Fig. 3 A, a conservative estimate indicates that 10 eq of proteolyzed RM may contain that quantity of SRα present in 0.5 eq of native RM (∼20-fold reduction). To assess the functional consequences of the proteolysis-dependent loss of SRα, the association of pPL86 RNCs with native and proteolyzed membranes was determined by flotation centrifugation (Fig. 3 B). A number of aspects of these experiments warrant mention. First, the experiments were performed with a truncated secretory precursor, pPL86. Previous studies have unequivocally demonstrated that this precursor remains in association with the ribosome and retains post-translational targeting and translocation competence (18Connolly T. Gilmore R. J. Cell Biol. 1986; 103: 2253-2261Crossref PubMed Scopus (115) Google Scholar, 19Nicchitta C.V. Blobel G. J. Cell Biol. 1989; 108: 789-795Crossref PubMed Scopus (53) Google Scholar). Thus, any functional SRα present in the proteolyzed RM fraction would be expected to be accessible to pPL86 RNCs and would display activity in the targeting assay. Second, as noted above, under standard reaction conditions, the total translation activity of the lysate-derived free ribosomes is approximately 10-fold higher than the membrane-bound ribosomes. To ensure that these experiments accurately depicted the activities of the free and membrane-bound ribosomes, the ribosome content of the reticulocyte lysate was titrated (by centrifugation and ribosome re-addition) such that identical quantities of precursor proteins were synthesized by either the free or membrane-bound ribosomes. Importantly, by so doing," @default.
• W2048423346 created "2016-06-24" @default.
• W2048423346 creator A5053140135 @default.
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• W2048423346 date "2000-10-01" @default.
• W2048423346 modified "2023-10-14" @default.
• W2048423346 title "Regulation of Ribosome Detachment from the Mammalian Endoplasmic Reticulum Membrane" @default.
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• W2048423346 doi "https://doi.org/10.1074/jbc.m005294200" @default.
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