Characterization of mitochondrial and cytoplasmic ribosomes from Paramecium aurelia

The ribosomes extracted from the mitochondria of the ciliate, Paramecium aurelia, have been shown to sediment at 80S in sucrose gradients. The cytoplasmic ribosomes also sediment at 80S but can be distinguished from their mitochondrial counterparts by a number of criteria. Lowering of the Mg++ concentration, addition of EDTA, or high KCl concentrations results in the dissociation of the cytoplasmic ribosomes into 60S and 40S subunits, whereas the mitochondrial ribosomes dissociate into a single sedimentation class at 55S. Furthermore, the relative sensitivity of the two types of ribosome to dissociating conditions can be distinguished. Electron microscopy of negatively stained 80S particles from both sources has also shown that the two types can be differentiated. The cytoplasmic particles show dimensions of 270 X 220 A whereas the mitochondrial particles are larger (330 X 240 A). In addition, there are several distinctive morphological features. The incorporation of [14C]leucine into nascent polypeptides associated with both mitochondrial and cytoplasmic ribosomes has been shown: the incorporation into cytoplasmic 80S particles is resistant to erythromycin and chloramphenicol but sensitive to cycloheximide, whereas incorporation into the mitochondrial particles is sensitive to erythromycin and chloramphenicol but resistant to cycloheximide.     

Small nuclear ribonucleoprotein antigens are absent from 10S translation inhibitory ribonucleoprotein but present in cytoplasmic messenger ribonucleoprotein and polysomes

A cytoplasmic 10S ribonucleoprotein particle (iRNP), which is isolated from chick embryonic muscle, is a potent inhibitor of mRNA translation in vitro and contains a 4S translation inhibitory RNA species (iRNA). The iRNP particle shows similarity in size to the small nuclear ribonucleoprotein (snRNP) particles. Certain autoimmune disease patients contain antibodies directed against snRNP antigenic determinants. The possibility that iRNP may be related to the small nuclear particles was tested by immunoreactivity with monospecific autoimmune antibodies to six antigenic determinants (Sm, RNP, PM-1, SS-A (Ro), SS-B (La), and Scl-70). By Ouchterlony immunodiffusion assays, the cytoplasmic 10S iRNP did not show any immunoreactivity. Also, a more sensitive hemagglutination inhibition assay for detecting Sm and RNP antigens failed to show reactivity with the 10S iRNP. Thus, the 10S iRNP particles are distinct from the similarly sized snRNP. However, free and polysomal messenger ribonucleoprotein (mRNP) particles and polysomes also isolated from chick embryonic muscle and analyzed by Ouchterlony immunodiffusion and hemagglutination inhibition for the presence of the antigenic determinants showed reactivity to Sm and RNP autoantibodies, but were not antigenic for the other four antibodies. Some of the Sm antigenic peptides of mRNP particles and polysomes were identical to those purified from calf thymus nuclear extract, as judged by Western blot analysis. The association of Sm with free and polysomal mRNP and polysomes suggests that Sm may be involved in some cytoplasmic aspects of mRNA metabolism, in addition to a nuclear function in mRNA processing.     

Study of the interaction of soluble and particle-bound nicotinamide adenine dinucleotide pyrophosphorylase with cytoplasmic ribosomes

The cytoplasm of cells infected with EMC virus contains new structures which possess activity of the nuclear enzyme NAD pyrophosphorylase [14]. An attempt was made to understand the mode of formation of these structures in the infected cell. It was found that soluble NAD pyrophosphorylase manifests a strong affinity for cytoplasmic ribosomes, sedimenting at 90S. When cytoplasmic ribosomes were dissociated to the 60S and 40S subunits, the enzyme was found to be adsorbed only to the 60S unit. In extracts of rat liver nuclei, NAD pyrophosphorylase is associated with 35S particles, composed mainly of protein and DNA. The bond between enzyme and particle is of a loose nature. When ribosomes are mixed with 35S nuclear particles, most of the enzyme activity is transferred from the nuclear particles to the ribosomes, thus forming particles with an average sedimentation coefficient of 90S. Similar structures are obtained when either soluble NAD pyrophosphorylase or 35S nuclear particles are mixed with preparations of cytoplasm isolated from non-infected cells. The results of these experiments suggest that the 90S cytoplasmic structures found in virus-infected cells could result from an association between either free or particle-bound NAD pyrophosphorylase with cytoplasmic ribosomes.     

Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1

Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.     

Comparative ultrastructure of selected oral streptococci: thin-sectioning and freeze-etching studies.

The ultrastructure of Streptococcus mutans, serotypes a-e, S. sanguis, S. mitis, and S. salivarius HHT, were examined by the techniques of thin-sectioning and freeze-etching. The cell walls varied in width between 15 and 46 nm and were covered with an electron-dense fibrillar or fuzz layer. The cytoplasmic membrane was in close association with numerous mesosomes which were, in turn, either closely associated or in contact with the bacterial chromosome. In freeze-etch replicas, the outermost layer of the cell wall was fibrous, and the cytoplasmic membrane was covered with particles composed of several subunits. Both particle-clusters and particle-free areas occurred on the surfaces of the cytoplasmic membrane, as well as a crystalline array in the ground plasm of the cell.     

Active regulator of SIRT1 is required for ribosome biogenesis and function

Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD⁺-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Characterization of mesosomes of Micrococcus luteus: isolation and properties of mesosomal ribosomes, and localization of penicillin-binding proteins in mesosomal membranes

Mesosomes were isolated and purified from Micrococcus luteus under hypertonic conditions throughout preparation processes. The purified mesosomal preparation was composed of closed tubules and vesicles. Electron-dense ribosome-like particles were observed within the isolated mesosomal vesicles by electron microscopy. The ribosome-like particles were isolated from the purified mesosomes by a procedure involving solubilization of the membranes with detergents followed by centrifugation on a linear density gradient of sucrose. The isolated particles have a sedimentation coefficient of 70S in the presence of 10 mM Mg2+, when Mg2+ concentration was lowered to 0.1 mM, the particles were dissociated into two sub-particles of 30S and 50S. The 70S particles had the same appearance as cytoplasmic 70S ribosome particles upon observations of negatively stained preparations. These findings indicate that mesosomal tubules contain ribosomes. The isolated mesosomal ribosomes had the ability for protein synthesis when polyuridylic acid-directed polyphenylalanine synthesis was assayed. The sensitivity of mesosomal ribosomes to inhibitors, chloramphenicol and streptomycin, for protein synthesis was significantly lower than that of both cytoplasmic and cytoplasmic membrane-bound ribosomes. In addition, three penicillin-binding proteins were detected in the mesosomal membranes. One of these was localized predominantly in the mesosomal membranes and the other two were distributed almost equally in both mesosomal and cytoplasmic membranes.     

Poliovirus morphogenesis. I. Identification of 80S dissociable particles and evidence for the artifactual production of procapsids.

The current model of poliovirus morphogenesis postulates a fundamental role for procapsid, 80S shells that, upon interaction with viral RNA and subsequent proteolytic cleavage, give rise to complete virus particles. Although 80S sedimenting particles can, indeed, be isolated from cytoplasmic extracts of infected cells, their physical properties differ from those reported for procapsids. Far from being stable structures, they can be dissociated by pH 8.5 and 0.1% sodium dodecyl sulfate into slower-sedimenting subunits. The reasons for this discrepancy were investigated, and two main modalities leading to the appearance of procapsids in vitro were identified. The first involves a temperature-mediated conversion of dissociable 80S particles into stable 80S procapsids, and the second involves the self-assembly of endogenous 14S subunits, also primed by an increase in the temperature of cytoplasmic extracts.     

Analysis of cytoplasmic 19 S ring-type particles in Drosophila which contain hsp 23 at normal growth temperature

Cytoplasmic 19 S particles were isolated from postpolysomal supernatants of 25 degrees C Drosophila embryos and culture cells. The particles were purified by salt extraction and sucrose gradient centrifugation. Electron microscopic investigation showed that the 19 S particles possess a ring-shaped morphology with an outer diameter of 12 nm and a hollow core of 3 nm. Biochemically the particles are characterized by a group of 16 polypeptides within the molecular weight range of 35 to 23 kDa, and small RNA molecules in the size range of 200 to 60 nucleotides. The RNP character of the particles is also shown by their buoyant density in Cs2SO4 of rho = 1.29 g/cm3 and their susceptibility to uv crosslinking and density in CsCl of rho = 1.38 g/cm3. Antibodies were raised against the proteins of the 19 S particles isolated from 25 degrees C cells and tested by immunoblotting after one- and two-dimensional gel-electrophoresis. Two of the antibodies raised cross react with the small heat-shock proteins hsp 28/27 and hsp 23. Comparative protease V8 cleavage of hsp 23 and the 23-kDa particle protein demonstrates that these two proteins are identical and that the small hsp of Drosophila must be a genuine part of the 19 S cytoplasmic ring-shaped complexes at normal growth temperature. The data support the idea of a general developmental role of some of the so-called heat-shock proteins.     

Immature small ribosomal subunits can engage in translation initiation in Saccharomyces cerevisiae

It is generally assumed that, in Saccharomyces cerevisiae, immature 40S ribosomal subunits are not competent for translation initiation. Here, we show by different approaches that, in wild‐type conditions, a portion of pre‐40S particles (pre‐SSU) associate with translating ribosomal complexes. When cytoplasmic 20S pre‐rRNA processing is impaired, as in Rio1p‐ or Nob1p‐depleted cells, a large part of pre‐SSUs is associated with translating ribosomes complexes. Loading of pre‐40S particles onto mRNAs presumably uses the canonical pathway as translation‐initiation factors interact with 20S pre‐rRNA. However, translation initiation is not required for 40S ribosomal subunit maturation. We also provide evidence suggesting that cytoplasmic 20S pre‐rRNAs that associate with translating complexes are turned over by the no go decay (NGD) pathway, a process known to degrade mRNAs on which ribosomes are stalled. We propose that the cytoplasmic fate of 20S pre‐rRNA is determined by the balance between pre‐SSU processing kinetics and sensing of ribosome‐like particles loaded onto mRNAs by the NGD machinery, which acts as an ultimate ribosome quality check point.     

Symplekin,a constitutive protein of karyo- and cytoplasmic particles involved in mRNA biogenesis in Xenopus laevis oocytes

Symplekin is a dual location protein that has been localized to the cytoplasmic plaques of tight junctions but also occurs in the form of interchromatin particles in the karyoplasm. Here we report the identification of two novel and major symplekin-containing protein complexes in both the karyo- and the cytoplasm of Xenopus laevis oocytes. Buffer-extractable fractions from the karyoplasm of stage IV-VI oocytes contain an 11S particle, prepared by immunoselection and sucrose gradient centrifugation, in which symplekin is associated with the subunits of the cleavage and polyadenylation specificity factor (CPSF). Moreover, in immunofluorescence microscopy nuclear symplekin colocalizes with protein CPSF-100 in the "Cajal bodies." However, symplekin is also found in cytoplasmic extracts of enucleated oocytes and egg extracts, where it occurs in 11S as well as in ca. 65S particles, again in association with CPSF-100. This suggests that, in X. laevis oocytes, symplekin is possibly involved in both processes, 3'-end processing of pre-mRNA in the nucleus and regulated polyadenylation in the cytoplasm. We discuss the possible occurrence of similar symplekin-containing particles involved in mRNA metabolism in the nucleus and cytoplasm of other kinds of cells, also in comparison with the nuclear forms of other dual location proteins in nuclei and cell junctions.     

ELECTRON MICROSCOPE STUDY OF MITOCHONDRIAL 60S AND CYTOPLASMIC 80S RIBOSOMES FROM LOCUSTA MIGRATORIA

Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl₂. Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of ~270 x 210 x 215 Å; cytoplasmic ribosomes measure ~295 x 245 x 255 Å. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is ~130 Å, while the diameter of cytoplasmic ribosomes is ~ 180–200 Å. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.     

The nucleoli of cultured human lymphocytes : I. Nucleolar morphology in relation to transformation and the DNA cycle

As the number of phases of the DNA cycle increases during the first 48 h of lymphocyte culture, there is a corresponding increase in the variety of nucleolar morphology. Nucleoli with the ‘resting’ ring-type distribution of RNP decline in frequency while first those with a uniform distribution of RNP, then those with a trabecular distribution increase. There is evidence that lymphocytes in S have trabecular nucleoli containing four nucleolini in the first half of S and from 5 to 8 nucleolini in late S. During G 2 the contents of the nucleolini and the body of the nucleolus appear to disperse. S and G2 nucleoli tend to be ‘irregular’ in shape. Some of the new nucleoli differentiating in post-telophase show processes of RNP from their surfaces, sometimes extending to the nuclear membrane or to other nucleoli. Whilst their condition is uniform or ring-type, their shape may vary between round, oval, lobed or elongated.     

Identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing RNA-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.     

Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.     

Proteome dynamics and proteome function of cardiac 19S proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.     

Human RioK3 is a novel component of cytoplasmic pre-40S pre-ribosomal particles

Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.     

Affinity labeling of the proteasome by a belactosin A derived inhibitor

Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facilitate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.