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The Fate of Membrane-bound Ribosomes Following the Termination of Protein Synthesis*

  • Robert M. Seiser
    Affiliations
    Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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  • Christopher V. Nicchitta
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell Biology, P. O. Box 3709, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-8948; Fax: 919-684-5481
    Affiliations
    Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant DK47897 (to C. V. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:October 27, 2000DOI:https://doi.org/10.1074/jbc.M004462200
      Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.
      ER
      endoplasmic reticulum
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      DMEM
      Dulbecco's modified Eagle's medium
      PAGE
      polyacrylamide gel electrophoresis
      The endoplasmic reticulum (ER)1 membrane, the site of nascent secretory and integral membrane protein translocation, contains an abundance of membrane-bound ribosomes. As is now well established, the association of biosynthetically active ribosomes with the ER membrane occurs through the activity of the signal recognition particle/signal recognition particle receptor targeting machinery (
      • Walter P.
      • Ibrahimi I.
      • Blobel G.
      ,
      • Meyer D.I.
      • Krause E.
      • Dobberstein B.
      ,
      • Gilmore R.
      • Blobel G.
      ). By this process, ribosome-nascent chain complexes bearing secretory or membrane protein precursors are recognized early in synthesis and are trafficked from the cytosol to the ER membrane (
      • Walter P.
      • Johnson A.E.
      ). Following the binding of the ribosome-nascent chain complexes to the resident translocon complex of the ER membrane, protein translation proceeds and nascent chains are translocated across or integrated into the ER membrane. Subsequently, the termination of protein synthesis is thought to elicit the release of the ribosomal subunits from the ER membrane to the free, cytoplasmic pool (
      • Blobel G.
      • Dobberstein B.
      ,
      • Mechler B.
      • Vassalli P.
      ). In the cytoplasmic pool, the ribosomal subunits are free to participate in the protein synthesis initiation sequence. Should they engage in the synthesis of a secretory or membrane protein precursor, targeting to the ER membrane again occurs, thus describing a cycle of ribosome binding and release.
      In the pioneering studies on ribosome-membrane interactions in the ER, it was observed that ribosomes bind asymmetrically, with the binding interaction being mediated entirely through the large subunits (
      • Sabatini D.D.
      • Tashiro Y.
      • Palade G.E.
      ,
      • Florendo N.T.
      ,
      • Baglioni C.
      • Bleiberg I.
      • Zauderer M.
      ,
      • Unwin P.N.
      ). Thus, if isolated ER microsomes were extracted with increasing concentrations of EDTA, the small ribosomal subunit was preferentially released from the membrane, whereas the large ribosomal subunit remained membrane-bound (
      • Sabatini D.D.
      • Tashiro Y.
      • Palade G.E.
      ,
      • Florendo N.T.
      ). Confirmation of the nature of ribosome binding to the ER was later obtained by direct ultrastructural analysis of intact cells, where it was observed that the large ribosomal subunit contains the site of membrane attachment (
      • Unwin P.N.
      ). On the basis of these observations, it may be predicted that the termination of protein synthesis by membrane-bound ribosomes would be accompanied by the regulated dissociation of large ribosomal subunits from the ER membrane.
      Do ribosomes cycle between a membrane-bound and free state? This question was first addressed in biosynthetic labeling and exchange studies and yielded differing and somewhat contradictory conclusions (
      • Baglioni C.
      • Bleiberg I.
      • Zauderer M.
      ,
      • Mechler B.
      • Vassalli P.
      ). In the study of Baglioni et al. (
      • Baglioni C.
      • Bleiberg I.
      • Zauderer M.
      ), it was proposed that large ribosomal subunits bind to the ER membrane prior to their assembly into polysomes. In contrast, Mechler and Vassalli (
      • Mechler B.
      • Vassalli P.
      ), using a similar isotope incorporation protocol, concluded that small and large ribosomal subunits enter the membrane-bound polysome fraction from the cytosolic pool at similar rates. Although these studies are in disagreement regarding the entry path for large subunits into membrane-bound polysomes, two points are evident. 1) The biosynthetic labeling and exchange kinetics of membrane-bound large subunits differ from those of membrane-bound small subunits. 2) Membrane-bound and cytosolic ribosome pools constitute a common population. However, because ribosome synthesis and assembly are relatively slow and complex processes, the resolution offered by ribosome biosynthetic labeling studies is not sufficient to analyze directly the temporal coupling between protein synthesis on membrane-bound ribosomes and ribosome exchange between free and bound ribosome pools.
      The purpose of this study was to investigate experimentally the compartmental fate of membrane-bound ribosomes following the termination of protein synthesis. Do ribosomes dissociate from the ER membrane coincident with the termination of protein synthesis? By using biochemical and morphometric methods in tissue culture cells, we report that in vivo termination yields the preferential release of ribosomal small subunits from the membrane, whereas the large ribosomal subunits remain predominantly in the bound state.

      DISCUSSION

      We report that the termination of protein synthesis on membrane-bound ribosomes in vivo results in the enhanced release of small ribosomal subunits from the membrane-bound pool; large ribosomal subunits remain in stable association with the ER membrane. Although these data do not support the proposal that the termination of protein synthesis on membrane-bound ribosomes yields the release of large and small ribosomal subunits into the free, cytoplasmic pool, they are consistent with prior data demonstrating that, in vitro, the release of nascent chains from membrane ribosomes results in the enhanced exchange of small but not large ribosomal subunits (
      • Borgese D.
      • Blobel G.
      • Sabatini D.D.
      ).
      From a historical perspective, the question of whether membrane-bound ribosomes participate in a translation-dependent exchange with free ribosomes closely followed the observation that free, cytosolic ribosomal subunits associate and dissociate coincident with the initiation and termination stages of translation (
      • Kaempfer R.
      • Meselson M.
      ,
      • Falvey A.K.
      • Staehelin T.
      ). Since it had been established that ribosomes were segregated between free and membrane-bound pools, studies were thus performed to determine whether such ribosome pools were kinetically exchangeable (
      • Mechler B.
      • Vassalli P.
      ,
      • Baglioni C.
      • Bleiberg I.
      • Zauderer M.
      ,
      • Mechler B.
      • Vassalli P.
      ,
      • Borgese D.
      • Blobel G.
      • Sabatini D.D.
      ,
      • Mechler B.
      • Vassalli P.
      ). Two experimental approaches were taken. In one approach, radioisotope incorporation studies were performed to determine the rate of entry of ribosomal subunits into the free and membrane-bound pools of intact cells. In the study of Baglioni et al. (
      • Baglioni C.
      • Bleiberg I.
      • Zauderer M.
      ), the primary conclusion was that large ribosomal subunits bind to the ER membrane, and polysome assembly occurs as small subunit initiation complexes joined with membrane-bound large subunits to yield translationally active 80 S ribosomes. This conclusion was disputed by Mechler and Vassalli (
      • Mechler B.
      • Vassalli P.
      ,
      • Mechler B.
      • Vassalli P.
      ,
      • Mechler B.
      • Vassalli P.
      ) in a detailed and rigorous series of investigations. These authors concluded that the kinetics of isotope incorporation into large ribosomal subunits of the free and bound ribosomal pools was such that the formation of 80 S ribosomes must occur in the free ribosome pool. Mechler and Vassalli (
      • Mechler B.
      • Vassalli P.
      ) did report, however, that the kinetics of membrane dissociation of large and small ribosomal subunits was different, with large ribosomal subunit release lagging that of small subunit release. In contrast to the above referenced studies, which were performed in intact cells, Borgeseet al. (
      • Borgese D.
      • Blobel G.
      • Sabatini D.D.
      ) examined ribosomal subunit exchange in vitro and reported that following the release of nascent chains, ribosomal subunit exchange was strictly limited to small subunits; large subunits remained in stable association with the microsomal membranes. Because it could not be unequivocally concluded that thein vitro system faithfully executed the termination reaction as seen in vivo, and it was known that free and membrane-bound ribosomal subunits were structurally and metabolically similar, conclusions regarding the physiological significance of an inexchangeable large ribosomal subunit pool were appropriately conservative (
      • Borgese D.
      • Blobel G.
      • Sabatini D.D.
      ).
      Although the isotope incorporation studies provided valuable data demonstrating that free and membrane-bound ribosomes existed in metabolic equilibrium, the experimental approach is hampered by a significant kinetic constraint. That is, the rates of ribosomal subunit biosynthesis and nuclear export are such that isotope incorporation rates must be followed for periods of hours (
      • Mechler B.
      • Vassalli P.
      ,
      • Baglioni C.
      • Bleiberg I.
      • Zauderer M.
      ,
      • Mechler B.
      • Vassalli P.
      ). Protein synthesis, however, occurs in the time frame of seconds to minutes, and thus the relevant exchange kinetics differ by 1–2 orders of magnitude. With this experimental limitation in mind, we re-examined the fate of membrane-bound ribosomes following the termination of protein synthesis through a combined morphometric and biochemical analysis of ribosome-membrane interactions in intact cells. The results of our studies best support the hypothesis that the termination of protein synthesis on membrane-bound ribosomes results in the free exchange of small ribosomal subunits, with the large ribosomal subunits remaining in stable association with the ER membrane. These data thus confirm and extend the conclusions obtained by Borgese et al. (
      • Borgese D.
      • Blobel G.
      • Sabatini D.D.
      ) and demonstrate that in the intact cell, large ribosomal subunit release from the ER membrane does not occur coincident with the termination of protein synthesis.
      What is the fate of membrane-bound large ribosomal subunits after termination? Should such subunits be competent for protein translation, it is likely that protein synthesis could be initiated on the endoplasmic reticulum membrane. Assuming this to be true, it then becomes necessary to determine whether membrane-bound ribosomes can select mRNA substrates and thus whether membrane-bound ribosomes can catalyze the synthesis of free, cytosolic proteins. Furthermore, as membrane-bound ribosomes are thought to reside in intimate association with the protein conducting channel component of the ER translocon, it is equally important that the compartmental fate of such translation products be determined. As it is known that membrane-bound large ribosomal subunits exchange with the cytoplasmic pool, it is essential that the mechanism of large subunit release be determined and the factors governing this release process be identified. Insights into these questions are presented in the accompanying manuscript (
      • Potter M.D.
      • Nicchitta C.V.
      ).

      Acknowledgments

      We gratefully acknowledge Drs. M. Reedy and J. Corless for access to electron microscopy equipment and supplies; and E. Worniallo, C. Lucaveche, and T. Zheng for excellent technical assistance. We thank M. Potter, R. Lerner, C. Rioja and other members of the laboratory for stimulating discussions. Portions of this study were performed with the electron microscopy facility of the Duke University Comprehensive Cancer Center.

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