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1.
Prosomes (proteasomes) of higher plants   总被引:3,自引:0,他引:3  
From different plant tissues such as tobacco (Nicotiana rustica), potato (Solanum tuberosum), and mung bean (Phaseolus radiatus), ring- or cylinder-shaped particles called prosomes were isolated by either sucrose gradient centrifugation or fast protein liquid chromatography (FPLC). These particles have a diameter of 12 to 14 nm and a length of 16 to 18 nm. They migrate under conditions of nondenaturing gel electrophoresis as one distinct band. Sedimentation coefficient and buoyant density in Cs2SO4 of the plant prosomes were determined by analytical ultracentrifugation to be approximately 23S and 1.23 g/cm3, respectively. The total molecular mass was estimated by gel filtration to be 650 kDa. Plant prosomes are composed of 12 to 15 proteins with molecular masses in the range of 24 to 35 kDa with isoelectric points of pH 5 to 7 as revealed by two-dimensional gel electrophoresis. The protein patterns of prosomes from the three different plant species are very similar. Polyclonal antisera against potato prosomes reacted in Western blots with prosomal proteins of all three plant species. They also bind to some prosomal proteins of animal species. Antisera against animal prosomes react with some proteins of plant prosomes. As shown by lectin blotting, plant prosomes are glycosylated carrying glucosyl- or mannosyl, and N-acetylgalactosaminyl residues. Prosomal preparations contain non-stoichiometric amounts of small RNA of about 80 kDa. These results suggest that plant prosomes are structurally and functionally homologous to prosomes of other eukaryotic cells.  相似文献   

2.
Viral messengers were used to select and purify prosomes and prosomal RNA from subribosomal fractions of HeLa cells and mouse erythroblasts. Adenovirus mRNA immobilized on oligo(dT)-cellulose and tobacco mosaic virus RNA (TMV) sedimenting in sucrose gradients associated strongly with prosomes at high salt conditions forming intermolecular RNA-RNA hybrids between prosomal RNA and viral RNA. Hybrid selection of small cytoplasmic RNAs with immobilized TMV-RNA revealed a RNA species migrating at the same position as prosomal RNA. The possible existence of a box-like sequence involved in hybridization will be discussed.  相似文献   

3.
4.
Prosomes were first described as being mRNA-associated RNP (ribonucleoprotein) particles and subcomponents of repressed mRNPs (messenger ribonucleoprotein). We show here that prosomes isolated from translationally inactive mRNP have a protease activity identical to that described by others for the multicatalytic proteinase complex (MCP, 'proteasome'). By RNase or non-ionic detergent treatment, the MCP activity associated with repressed non-globin mRNP from avian erythroblasts, sedimenting at 35 S, could be quantitatively shifted on sucrose gradients to the 19-S sedimentation zone characteristic of prosomes, which were identified by monoclonal antibodies. The presence of small RNA in the enzymatic complex was shown by immunoprecipitation of the protease activity out of dissociated mRNP using a mixture of anti-prosome monoclonal antibodies; a set of small RNAs 80-120 nucleotides long was isolated from the immunoprecipitate. Furthermore, on CsCl gradients, colocalisation of the MCP activity with prosomal proteins and prosomal RNA was found, and no difference in the prosomal RNA pattern was observed whether the particles were fixed or not prior to centrifugation. These data indicate that the MCP activity is a property of prosomes, shown to be in part RNP and subcomplexes of in vivo untranslated mRNP. A hypothesis for the role of the prosome-MCP particles in maintaining homeostasis of specific protein levels is proposed.  相似文献   

5.
Prosomes. Ubiquity and inter-species structural variation   总被引:13,自引:0,他引:13  
The "prosomes", a novel type of ubiquitous ribonucleoprotein particle of extraordinary stability and of defined electron microscopical structure, have been characterized in several cell types and species. Identified as a 19 S sub-component of free mRNA-protein complexes, including globin and other repressed mRNA, in the cytoplasm of duck, mouse and HeLa cells, they were previously found to inhibit protein synthesis in vitro. In all cells studied, electron microscopy shows an identical, seemingly ring-like but rather raspberry-shaped particle of 12 nm diameter, resistant to EDTA and 1% (w/v) Sarkosyl. Two-dimensional electrophoretic analysis of prosomal proteins shows a characteristic pattern in the 19,000 to 35,000 Mr range of pI 4 to 7, with an additional 56,000 Mr component specific to avian species. The prosomes found in globin mRNA-protein complexes contain about 25 protein components, 16 of which have identical molecular weight and pI values in duck and mouse, and which are also found in the prosomes of the heterogeneous free mRNPs of HeLa cells. Seral and monoclonal antibodies raised in mice against the prosomes of duck erythroblasts cross-react with some of the proteins of the mouse and HeLa cell particles. Prosomes isolated from duck and mouse globin mRNP, both contain small cytoplasmic RNAs of 70 to 90 nucleotides, which represent about 15% of the particle mass. The molecular weight and the 3'-terminal oligonucleotide of each one of these small cytoplasmic RNAs are identical in the two animal species; fingerprints of their oligonucleotides generated by RNase T1 show that more than 80% of spots are identical. In contrast, the prosomes of HeLa cells, associated with a large population of repressed mRNA, contain at least 12 small cytoplasmic RNA species. All prosomal RNAs tested so far hybridize to mRNA. The data available indicate that prosomes constitute a novel class of ubiquitous cellular ribonucleoprotein complexes, present in the nucleus and cytoplasm that, in its structural variations shown here, reflects function and species.  相似文献   

6.
Prosomes, small cytoplasmic particles of mouse erythroblasts were found to contain low molecular weight RNA molecules in the range of 80 nucleotides. Nuclease digestion of prosomes suggests that prosomal proteins cover and protect almost the whole length of their RNA(s). Our results demonstrate clearly that RNA is an intrinsic component of prosomes.  相似文献   

7.
Here we demonstrate that the epidermal growth factor (EGF) induces association of prosomes (20S-proteasomes) with its receptor in A-431 cells. Additionally, ligand-dependent association of ribonucleoprotein particles (alpha-RNP), containing small ALU-like RNA, with the EGF receptor was demonstrated. A suggestion has been put forward on the involvement of prosomes and alpha-RNP in the EGF signal transmission to different stages of gene expression.  相似文献   

8.
In heat-shocked tomato cell cultures, cytoplasmic heat shock granules (HSGs) are tightly associated with a specific subset of mRNAs coding mainly for the untranslated control proteins. This messenger ribonucleoprotein complex was banded in a CsCl gradient after fixation with formaldehyde (approximately 1.30 g/cm3). It contains all the heat shock proteins and most of the RNA applied to the gradient. During heat shock, a reversible aggregation of HSGs from 15S precursor particles can be shown. These pre-HSGs are not identical to the 19S plant prosomes. Ultrastructural analysis supports the ribonucleoprotein nature of HSGs and their composition of approximately 10-nm precursor particles. A model summarizes our results. It gives a reasonable explanation for the striking conservation of untranslated mRNAs during heat shock and may apply also to animal cells.  相似文献   

9.
Prosomes (20S proteasomes) constitute the catalytic core of the 26S proteasomes, but were first observed as factors associated with unstranslated mRNA. Recently, their RNase activity was discovered together with the fact that their proteolytic function is dispensable in adapted human cells. By indirect immunofluorescence using monoclonal antibodies, we demonstrate as a general phenomenon, regular intercalation of specific types of prosomes into the sarcomeric structure of all types of striated muscle. Surprisingly, in cultured smooth muscle cells without sarcomeric organization, some prosomes also form regular striations in extended projections of cytoplasmic regions. The significance of their sarcomeric distribution is not understood as yet, but the pattern we observe is very similar to that shown by others for muscle-specific mRNAs, identified by in situ hybridization, and that of the cognate proteins. A role of prosomes in the cotranslational assembly of the myofibrillar proteins is suggested, since prosomes organize into pseudo-sarcomeric patterns prior to formation de novo of the actin-myosin arrangement.  相似文献   

10.
PROSOMES are a novel class of small RNP particles of uniform morphology, but of variable RNA (pRNA) and protein composition (about 650 000 MW; 12 nm diameter in the EM). They were discovered as subcomplexes of free mRNP, tightly attached to inactive mRNA in the cytoplasm. The pRNAs hybridize stably to mRNA. Prosomes associate in vitro to mRNA and inhibit cell free protein synthesis inducing an mRNA structure unable to interact with ribosomes. Many types of prosomes were observed. The individual particle is made up by a variable combination of about 20 characteristic proteins and one or several pRNA. Some prosomal proteins are glycosylated, phosphorylated and, possibly, ADP-ribosylated and are highly conserved in evolution whilst others vary with the species and the mRNA population they are associated to. A protease activity was found associated to prosomes.The function(s) of the prosomes is(are) still unknown. The differential inhibition of in vitro protein synthesis points to a capacity to recognize mRNA and to keep it in an inactive state. The observation with the aid of monoclonal antibodies (pMABs) that prosomes and thus mRNP are attached to the intermediate filaments (IF) raises the question if one of the functions of the IF might be in the topological distribution of mRNA within the cell. Similar to the cytokeratin fibers, the prosome networks bridge neighboring cells at specific positions. — The nucleus also contains some prosomal antigens, located on chromosomes and on the nuclear matrix. Their presence and distribution in the cell compartments varies with the cell type and the prosomal antigen probed.Oocytes contain large amounts of prosomes. In embryonic development, the synthesis of individual prosomal proteins starts progressively after the blastula stage and resumes fully in gastrulation only; cleavage and blastula stage prosomes are thus of maternal origin. The nucleo-cytoplasmic distribution of prosomal antigens changes in embryos, with the stage of development and type of differentiation. In human tissues specific patterns of prosomal antigens were found in function of cell type and differentiation.In view of these data, the hypothesis may be formulated that prosomes are a population of mRNA-linked RNP which includes particles of varying individual composition and hence specificity. Attached to IF sub-networks, specific types of prosomes might accompany families of mRNA in function of the physiological state and the specialisation of given differentiated cell types. The cell-type specific organisation of the IF networks might be related to the messenger RNA complement of a given cell, and to its status of gene expression. The prosomes might thus have a function in controlling the transport, distribution and control of activity of specific mRNAs in the cell.  相似文献   

11.
The core of the 26S proteasome, the 20S prosome, is a highly organized multi-protein complex found in large amount in malignant cells. Differentiation of several cell lines, including the monoblastic U937 and the lymphoblastoid CCRF-CEM, is accompanied by a general decrease in the prosome concentration when phorbol-myrirtic-acetate (PMA) and retinoic acid plus dihydroxyvitamine D3 (RA+VD) are used. Incubation of U937 cells for three days with PMA or RA+VD causes differentiation, but the resulting patterns of prosome labeling in the cell and on the plasma membrane are not the same. In contrast, the same kind of prosome changes occur in U937 and CCRF-CEM cells when PMA is used as inducer. The intracellular distribution of prosomes is also linked to malignancy and differentiation. Prosomes are found in the nucleus and the cytoplasm of cancer cells; and treatment with RA+VD decreases the prosomes in the nucleus whereas PMA causes various prosome proteins changes. These results indicate that prosomes are important in cell regulation and in the expression of malignancy.  相似文献   

12.
T Kiss  C Marshallsay    W Filipowicz 《The EMBO journal》1992,11(10):3737-3746
Mammalian MRP (for mitochondrial RNA processing) RNA, also known as 7-2 RNA, is a nuclear encoded small RNA which has been reported to function in two different cellular compartments: in the mitochondria and in the nucleus. The ribonucleoprotein particle which contains the 7-2/MRP RNA, called RNase MRP, has ribonucleolytic activity and shares some structural similarity with RNase P. It has been proposed that in mitochondria, the RNase MRP is responsible for endonucleolytic cleavage of primer RNA during DNA replication. We have characterized the gene and cDNAs encoding 7-2/MRP-like RNA in Arabidopsis and tobacco, and found that in plants this RNA is enriched in nucleoli but is undetectable in purified mitochondria isolated from tobacco leaves or cells grown in suspension. In glycerol gradients tobacco 7-2/MRP RNA cosediments with large approximately 80S structures possibly representing ribosomal precursors. Fractionation of HeLa cells has also revealed that 7-2/MRP resides in the nucleolus and that most of it is associated with complexes sedimenting at approximately 80S, similar to those containing the U3 nucleolar RNA which is known to participate in pre-rRNA processing. These results indicate that the 7-2/MRP ribonucleoparticle may be involved in ribosome biogenesis, in both plant and mammalian cells.  相似文献   

13.
Prosomes are small ribonucleoprotein (RNP) particles of unique morphology in the electron microscope but of variable protein and RNA composition, depending on the differentiation state of the cells studied. They were initially observed as subcomplexes of untranslated mRNP. In previous studies, we found that prosomes are associated to the intermediate filaments (IF) of cytokeratin type in HeLa and PtK1 cells. Here we have studied in detail the association of prosomal antigens with the IF networks in PtK1 cells. Contrary to our earlier conclusions, in these cells the vimentin fibers also carry prosomes which, thus, distribute in between the two types of networks. During the selective collapse of the IF induced by acrylamide, and upon recovery after the withdrawal of the drug, no dissociation of the prosome and IF networks of cytokeratin- and vimentin-type could be observed. These data show that even in a dynamic situation, prosome and IF antigens do not dissociate, indicating strongly that they are located on one and the same structure. Furthermore, the differential distribution of specific prosomal antigens between both types of intermediate filament networks indicates that prosomes do not ubiquitously populate the intermediate filaments but occupy subnetworks of either vimentin or cytokeratin type.  相似文献   

14.
We have examined the accessibility to diethylpyrocarbonate of spinach chloroplast 4.5S ribosomal RNA when free and when it is part of the ribosomal structure. The modifications in free 4.5S RNA were found mostly in single-stranded regions of the secondary structure model proposed in our previous paper (Kumagai, I. et al. (1982) J.B.C. 257, 12924-28): adenines at positions 17, 19, 33, 36, 54, 55, 60, 64, 68, 72, 77, 86 and 87 were identified as the reactive residues. On the other hand, in 4.5S RNA in 70S ribosomes or 50S subunits, adenine 33 was exclusively modified, and its reactivity was much higher than in free 4.5S RNA. This highly accessible A33 of spinach 4.5S RNA is located within a characteristic seven nucleotide sequence, which is found in the 4.5S rRNAs from spinach, tobacco and a fern but deleted in 4.5S RNAs from maize and wheat.  相似文献   

15.
Duck erythroblasts prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo-)electron microscopy. A molecular weight of approximately 720,000 +/- 50,000, a radius of gyration of 64 +/- 2 A and a hydrodynamic radius of approximately 86 A were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 A, a height of 170 A and a diameter of 40 A for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.  相似文献   

16.
Prosomes and multicatalytic proteinases were purified from rabbit erythrocyte lysates and were analysed to determine their relationship. During purification by sucrose density gradient centrifugation using low salt buffer, they sedimented at 20–26S. Upon further purification, using high salt buffer, prosomes were recovered as 20S complexes as determined by their characteristic polypeptide pattern. Interestingly, both the 26S and 20S components had protease activity. Therefore, in order to determine their relationship with the multicatalytic proteinases, which are reported to contain a similar set of polypeptides, highly pure prosomes and the multicatalytic proteinases were analysed. Both 20S prosomes and multicatalytic proteinases showed protease activity and also had identical protein subunits of molecular weight ranging from 21 kDa to 35 kDa. Among these, at least two were immunologically identical as determined by Western blot using two monoclonal antibodies prepared against duck prosomes. Furthermore, protease activities of both components were inhibited almost to the same extent by an endogenous inhibitor specific for high molecular weight proteases and calpain. These results thus establish that the 20S prosomes and multicatalytic proteinases are identical, and suggest further that proteolytic activity could be the principal function of prosomes.  相似文献   

17.
Prosomes were originally identified as 20S particles associated with untranslated mRNA; they also constitute the core of the 26S proteasomes. The cellular distribution of three types of prosomes characterized by the presence of subunits with molecular masses of 23, 27, and 30 kDa was analyzed using an immunocytochemical approach on cultured chicken erythroblasts. The prosomes containing the p27K and p30K subunits were found in diffuse distribution in both nuclei and cytoplasm. In contrast, the prosomes containing the p23K subunit, although relatively rare in the nuclear space, were found concentrated in one or two large spots. Using in situ hybridization with an alpha(A)-globin gene-specific riboprobe we found that the p23K-type prosomes colocalize in the nucleus with centers of globin (pre-)mRNA processing, and of mRNA accumulation in the cytoplasm. This result suggests there is local coincidence of specific-type prosome function with processing and, possibly, transport of a particular kind of (pre-)mRNA.  相似文献   

18.
Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.  相似文献   

19.
Several fractions of RNA prepared from the post-ribosomal cytosol of sheep lymphoid cells were found to include messenger-like RNA as defined by the following criteria: a, template activity, i.e. the ability to promote the incorporation of radioactive amino acids into protein in cell-free protein-synthesising systems derived from wheat embryos or ascites tumour cells; b, a low magnesium optimum (1-2.5 mM) for template activity which is characteristic of many natural mRNAs; c, sensitivity of the template response to aurintricarboxylic acid, a specific inhibitor of the initiation of protein synthesis. The lymphoid post-ribosomal RNA fractions, however, were translated less efficiently than were rabbit reticulocyte globin mRNA or tobacco mosaic viral (TMV) RNA; no explanation for this relatively poor template activity was found. The major fraction of messenger-like RNA had an average sedimentation coefficient of 12 S; this fraction directed the translation of several discrete polypeptides in the molecular weight range 10 000-25 000. On average the products of 12 S RNA-directed protein synthesis appeared lysine rich compared with TMV RNA-directed products. It is suggested that the apparent pool of uncommitted mRNA in resting lymphocytes may be utilised during the early stages of lymphocyte activation, and that the mRNAs could be stored in forms similar to those evident in other dormant tissues.  相似文献   

20.
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