TRANSFER OF PROTEINS ACROSS MEMBRANES I. Presence of Proteolytically Processed and Unprocessed Nascent Immunoglobulin Light Chains On Membrane-Bound Ribosomes of Murine Myeloma (original) (raw)
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The Journal of Cell Biology, 1975
The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCI procedure can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added lgG light chain mRNA so as to segregate the translation product in a proteolysis-resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed.
The Journal of Cell Biology, 1975
The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [aH]uridine pulse. Both 40S and 60S subunits enter free and membranebound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits, At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membranebound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.
The Journal of cell biology, 1981
Immunoglobulin heavy (Ig H) and light (Ig L) chain mRNA molecules have been released from the endoplasmic reticulum (ER) membranes as free (F) mRNP particles when MOPC 21 (P3K) mouse myeloma cells are exposed to a hypertonic initiation block (HIB). The subsequent fate of these mRNA sequences has been examined when the cells are returned to normal growth medium. Upon return to isotonicity, all previously translated mRNA molecules reassociate with ribosomes and form functional polysomes. Ig H mRNA is found incorporated first into F polysomes and then into membrane-bound (MB) polysomes. Kinetic studies indicate that the time of passage of Ig H mRNA in F polysomes is approximately 30 s, during which a nascent polypeptide chain of approximately 80 amino acids would have been completed. When the rate of polypeptide elongation is depressed with emetine during the recovery from HIB, both Ig H and L mRNA molecules accumulate in small F polysomes. These results indicate that the formation of ...
The Journal of Cell Biology, 1975
Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [ssS]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes.
Dissociation of Single Ribosomes as a Preliminary Step for Their Participation in Protein Synthesis
European Journal of Biochemistry, 1973
32P-labelled derived 40-S ribosomal subunits and 80-S monoribosomes entered the polyribosome fraction of a rabbit-reticulocyte lysate active in protein synthesis by a process that was dependent on time, energy and temperature. Both 40-S and 60-S subparticles from the labelled monoribosome entered the polyribosomes a t the same rate. The addition of excess unlabelled 40-S subparticles to the cell-free system containing labelled monoribosomes increased the ratio of 6 0 3 to 40-S-subparticle radioactivity in the polyribosome fraction. This indicated a competition between added unlabelled and monoribosome-derived 40-5 subparticles for entry into polyribosomes. From the evaluation of the translation time of mRNA, of the rate of monoribosomepolyribosome exchange, and of the equilibrium distribution of the cycling labelled monoribosomes and unlabelled subparticles with polyribosomes it is concluded that the dissociation of single ribosomes into subunits is an obligatory step for their entry into the poIyribosoma1 fraction and that this process is coupled with initiation of mRNA translation.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1988
The treatment of total endoplasmic reticulum membranes of mouse plasmacytoma cells with EDTA resulted in an abolition of the heavy rough (HR) subfraction, while there was a large increase in smooth (S) membranes. When HR and light rough (LR) endoplasmic reticuhim membranes were treated individually with EDTA and re-centrifuged on discontinuous sucrose gradients it was observed that HR were converted into S membranes, i.e. membranes virtually devoid of ribosomes. LR membranes were not affected to the same extent but there was a shift to a somewhat lower density. A quantitation of ribosomes released by EDTA showed that 95% of 60 S and 72% of 40 S subunits were removed from HR membranes while for LR membranes the corresponding values were 8.5 and 22.6% respectively. Ratios of radioactivity to absorbance at 260 nm calculated for 40 S and 60 S subunits isolated from HR and LR membranes show that 60 S subunits from LR membranes, in contrast to those from HR membranes, equilibrate only slowly with the free pool of ribosomal subunits. The results indicate that the ribosomes associated with HR membranes are 'loosely bound' and those with LR membranes 'tightly bound'. When poly(A)-containing mRNA isolated from HR and LR membranes was translated in vitro and the products analysed for light-chain immunoglobulin content, it was found that the HR fraction was enriched in light-chain mRNA.
Ribosomes and protein synthesis
2016
Let us start at the very beginning. Between 1897 and 1899, G. Gamier, in France, published elegant microscope studies describing a basophilic component ofthe cytoplasm of glandular cells (1). Because of what he thought its role might be in the elaboration and transformation of secretory products, he gave a Greek name to these concepts-ergastoplasm (work plasm). Gamier's research was extended by others-particularly A. Prenant, R. R. Bensley, and A. Matthews-to include other cell types, so that by the early part of this century ergastoplasm came to be a generally accepted term for a specific basophilic area of the cytoplasm. These early studies are extensively reviewed by F. Haguenau (1). The next major advance was to show that basophilia was due to RNA: in 1933, J. Brachet used RNase (2) ; in 1939, T. Caspersson used ultraviolet spectrophotometry (3); in 1943, J. N. Davidson and C. Waymouth used chemical methods (4). The high correlation which was shown between the amount ofRNA in various cells and the postulated protein-synthesizing capacity of those cells led Caspersson in 1941 (5) and Brachet in 1942 (6) to proclaim the importance of RNA in the process of protein synthesis. As can be imagined, this conjecture spurred many scientists in the next decade to try to answerthree questions. In what form was this cytoplasmic RNA? Did it really have a role in protein synthesis? Ifso, what was the role? Various methods were used : extraction and chemical procedures; extraction and physicochemical procedures, such as ultracentrifugation; and, because electron microscopy was becoming more and more refined, visualization. We now know that the RNA is in the form of ribosomes, and that the proteins of the ribosomes are involved in the many individual steps of protein synthesis; however, the function of ribosomal RNA is still elusive. The intensity of the research in the 1940s is caught very well in Haguenau's chapter on the visualization aspect (1) and in Magasanik's 1955 monograph (7) on the extraction and chemical properties of what were then called "pentose nucleoprotein." Confusion abounded, in good part due to the terminologies developed for the different techniques, such as Gamier's ergastoplasm, K. Porter's "endoplasmic reticulum" (8), A. Claude's "microsome" fraction (9), G. Palade's "small particulate component" (10), and the "nucleoprotein" preparations or particles discovered by various workers. The last are re
The Journal of cell biology, 1981
The subcellular distribution of the most abundant mRNA sequences, particularly those of the immunoglobulin heavy (Ig H) and light (IG L) chain mRNA sequences, of MOPC 21 (P3K) mouse myeloma cells has been examined by translating the mRNA of various subcellular fractions in a messenger-dependent reticulocyte lysate (MDL) and by identifying Ig products with the use of a specific antiserum. Analyses of the distribution of the mRNA template activity and the translation products by SDS polyacrylamide gel electrophoresis reveal that approximately 85% of the mRNA present in the free ribosomal fraction is incorporated into polysomes and that the remainder is present as mRNP particles. On the endoplasmic reticulum (ER) the mRNA is found entirely in polysomes. In general, the size class of free (F) and membrane-bound (MB) polysomes corresponds to the size of their translation products. Thus, mRNAs coding Ig H (5.0 x 10(5) daltons in size) and Ig L (2.5 x 10(5) daltons in size) are incorporate...
Isolation of ribosomes and assay systems for testing the translational apparatus have been part of the experimental routine of many laboratories for more than two decades, and excellent collections of the methods have been published previously, including two books in this series. However, a number of methods have been gradually and continuously improved and optimized. Major developments have included the improvement of ribosome isolation procedures, the establishment of highly efficient assay systems, and the application of heteropolymeric mRNA. The protocols in this chapter are concerned with the isolation of polysomes, ribosomes, and ribosomal subunits from prokaryotic and eukaryotic sources, as well as test systems for both total protein synthesis and single ribosomal functions, taking into account the recent developments.