Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria

Cultures of whole fetal rat sensory ganglia which had matured and myelinated in culture were treated for 1-3 h with a pulse of 0.2% trypsin. The tissue was observed during the period of treatment and during subsequent weeks using both light and electron microscopy. Within minutes after trypsin addition the matrix of the culture was altered and the nerve fascicles loosened. Progressive changes included the retraction of Schwann cell processes from the nodal region the detachment of the myelin-related paranodal Schwann cell loops from the axon, and lengthening of the nodal region as the axon was bared. The retraction of myelin from nodal stabilized several hours after trypsin withdrawal. Breakdown of the altered myelin segments was rare. There were no discernable changes in neurons or their processes after this exposure to trypsin. The partial repair which occured over a period of several weeks included the reattachment of paranodal Schwann cell loops to the axolemma and the insertion of new myelin segments where a substantial length of axolemma had been bared. The significance of these observations to the characterization of the Schwann cell-axolemmal junctions on myelinated nerve fibers is discussed. The dramatic degree of myelin change that can occur without concomitant myelin breakdown is particularly noted, as is the observation that these altered myelin segments are, in part, repaired.     

The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes

The DEAD-box helicase DDX3 has suggested functions in innate immunity, mRNA translocation and translation, and it participates in the propagation of assorted viruses. Exploring initially the role of DDX3 in the life cycle of hepatitis C virus, we observed the protein to be involved in translation directed by different viral internal ribosomal entry sites. Extension of these studies revealed a general supportive role of DDX3 in translation initiation. DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes. DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes. Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes.     

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations--implications for the study of ribosome dynamics

Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg(2+) is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg(2+) concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg(2+) that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg(2+) concentrations. In low Mg(2+) concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.     

Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography

We investigated the enzyme cytochemical localization of sarcosine oxidase (SOX) in the liver and kidney of several mammals using a cerium technique. First we measured the enzyme activities in the liver and kidney of several mammals and in several organs of mice. The highest activity was found in the Chinese hamster, followed by the mouse. Therefore, we used hamster and mouse tissues for enzyme cytochemistry. The liver and kidneys were fixed by perfusion with various concentrations of glutaraldehyde for 10 min. Tissue slices were incubated in reaction medium consisting of 50 mM TRIS-maleate buffer (pH 7.8), 9 mM sodium azide, 9.8 mM sarcosine, 25 microM FAD, 2 mM cerium chloride, 0.002% saponin, and 0.003% Triton X-100 for 0.5-8 h at 37 degrees C. Optimum staining reaction was obtained in tissues fixed with 0.2% glutaraldehyde, followed by incubation for 2-4 h. Electron-dense reaction products were present exclusively in peroxisomes. Within the peroxisomes strong reactions were observed in the matrix subjacent to the limiting membrane decreasing toward the center. The staining reaction was completely inhibited by 2 mM N-bromosuccinimide. These results indicated that SOX is a peroxisomal enzyme and that the enzyme might be associated with the peroxisomal membrane.     

Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840-1849 of 18S rRNA, but in the complexes formed with participation of Phe-TPHKPhe (where the G residue carrying the arylazido group occupied position-3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position-3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.     

Vigilin is co-localized with 80S ribosomes and binds to the ribosomal complex through its C-terminal domain

The biological relevance of vigilin a ubiquitous multi (KH)-domain protein is still barely understood. Investigations over the last years, however, provided evidence for a possible involvement of vigilin in the nucleo-cytoplasmic transport of tRNA and in the subsequent association of tRNA with ribosomes. We therefore investigated the potential association of vigilin with 80S ribosomes. Immunostaining, gel filtration, westernblot analysis of polyribosomes and high salt treatment of 80S ribosomes isolated from fresh human placenta were applied to analyze the possible association of vigilin with ribosomes. Overlay assays were performed to examine whether vigilin is capable of binding to ribosomal proteins. Immunostaining of HEp-2 cells, gel filtration of a cytoplasmic extract of HEp-2 cells and westernblot analysis of isolated 80S ribosomes clearly demonstrate that vigilin is bond to the ribosomal complex. Vigilin detaches from the ribosomal complex under the influence of high salt concentrations. We present data that radioactively labeled human vigilin interacts directly with a subset of ribosomal proteins from both subunits. We were able to narrow down the putative binding region to the C-terminal domain by using vigilin mutant constructs. Therefore our results provide strong evidence that vigilin is bond to the ribosomal complex and underline the hypothesis that vigilin might be involved in the link between tRNA-export and the channeled tRNA-cycle on ribosomes.     

Exploring protein import pores of cellular organelles at the single molecule level using the planar lipid bilayer technique

Proteins of living cells carry out their specialized functions within various subcellular membranes or aqueous spaces. Approximately half of all the proteins of a typical cell are transported into or across membranes. Targeting and transport to their correct subcellular destinations are essential steps in protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Virtually all proteins of the endosymbiotic organelles, chloroplasts and mitochondria, are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic and biochemical techniques led to rather detailed knowledge on the subunit composition of the various protein transport complexes which carry out the membrane transport of the preproteins. Conclusive concepts on targeting and cytosolic transport of polypeptides emerged, while still few details on the molecular nature and mechanisms of the channel moieties of protein translocation complexes have been achieved. In this paper we will describe the history of how the individual subunits forming the channel pores of the chloroplast, mitochondrial and endoplasmic reticulum protein import machineries were identified and characterized by single channel electrophysiological techniques in planar bilayers. We will also highlight recent developments in the exploration of the molecular properties of protein translocating channels and the regulation of the diverse protein translocation systems using the planar bilayer technique.     

Immunocytochemical localization of GABAergic neurones at the electron microscopical level

Summary Antibodies prepared to purified brain glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotrasmitter, -aminobutyric, acid (GABA), have been utilized with an unlabelled antibody method to localize GABAergic neurones in both light and electron microscopic preparations. A modification of Sternberger's peroxidase-antiperoxidase (PAP) complex is used to localize the site of anti-GAD binding, and the PAP complex is visualized with diaminobenzidine and H₂O₂. The reaction product is visible in both the light and electron microscopes. The ability to localize and identify labelled profiles in the electron microscope provides more functional information than light microscopical preparations. For example, the GAD-positive reaction product occurs mostly in association with synaptic vesicles within axon terminats, and this localization indicates the importance of GAD for the packaging and storage of GABA. The somata and dendrites of neurones giving rise to these terminals are visualized in colchicine-injected material. The GABAergic neurones form axo-somatic, axo-dendritic, axo-axonal and dendro-dendritic synapses in various regions of the rat central nervous system. Pretreatments of animals with anterograde degeneration have shown the significance of some of the GABAergic terminals that form axo-axonal synapses in the spinal cord.An many brain regions, such as the cerebral cortex, hippocampus and olfactory bulb, virtually all of the GABAergic synapses are derived from local circuit neurones. In other regions such as the cerebellum and neostriatum, the GABAergic terminals are derived from both local circuit neurones and the local axon collaterals of projection neurones that have their somata within these regions. A third type of configuration of GABAergic terminals occurs in the globus pallidus and substantia nigra where these terminals are derived from distant brain regions, axon collaterals of projection neurones and from local circuit neurones. Together, these results indicate the complex organization of the GABAergic system of the brain that has been vividly revealed with electron in croscopical immunocytochemistry.     

Structure of the mammalian 80S ribosome at 8.7 A resolution

In this paper, we present a structure of the mammalian ribosome determined at approximately 8.7 A resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for approximately 20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.     

Molecular architecture of adult locust cuticle at the electron microscope level

It is shown that locust adult endocuticle consists of a daily alternation of two types of chitin-protein architecture: (i) non-lamellate day layers with microfibrils oriented in a preferred direction, traversed by pore canals whose shape resembles an untwisted ribbon, (ii) lamellate night layers with helicoidally oriented microfibrils traversed by pore canals shaped like regularly twisted ribbons. Uncoupling the circadian clock which normally controls the timing of these two types leads to growth of cuticles which are organized like one or the other throughout. We can thus experimentally change the architecture of the microfibrils which in turn changes the pore canals.     

Localization of proteins S1, S2, S16 and S23 on the surface of small subunits of rat liver ribosomes by immune electron microscopy

Summary Ribosomal proteins S1, S2, S16 and S23 were localized on the surface of the small subunit (40S) of rat liver ribosomes by immune electron microscopy. Antibodies against the single proteins were raised in rabbits and chicken and purified by affinity chromatography. 40S-IgG-40S complexes were obtained by incubation of 40S subunits with non-crossreacting antibodies specific for each of the four proteins and subsequent sucrose density gradient centrifugation. The location of the proteins was determined by means of antibody binding sites visualized in negative contrast in the electron microscope. The four investigated proteins are mainly located in the head region of the small subunit. Exposed antigenic determinants of proteins S1 and S2 were found to be located at different sites of the small subunit whereas proteins S16 and S23 were mapped in a limited region only.S2,S3,S17,S21 according to the new nomenclature (McConkey et al., 1979)     

Localization of somatostatin receptors at the light and electron microscopial level by using antibodies raised against fusion proteins

Somatostatin mediates its multiple biological effects via specific plasma membrane receptors belonging to the family of G-protein coupled receptors with seven putative membrane-spanning domains. Five somatostatin receptor subtypes (sst1-sst5) have been cloned in human, mouse, and rat. We have raised specific antibodies against the five human somatostatin receptors by using the fusion protein technique. DNA sequences encoding C-terminal parts of the somatostatin receptors were inserted into a pGEX-2T plasmid vector. E. coli bacteria were transformed with the recombinant plasmid and fusion proteins were expressed and purified using the glutathione S-transferase Gene Fusion System. The fusion proteins were emulsified with Freund's complete adjuvant and polyclonal antibodies were raised in rabbits. The antisera were tested for specificity in Western blot analysis of membrane preparations from cell lines expressing the receptors and in membrane preparations of brain tissues. The receptors were visualized at the light microscopical level in paraformaldehyde fixed tissue sections by use of biotin labelled secondary antibodies as well as by amplification with biotinylated tyramide. The final step in the immunohistochemical visualization of the receptors was done by both peroxidase labelled streptavidin/biotin and different fluorophores. At the electron microscopical level, some of the receptors could be visualized in tissues fixed with a combination of paraformaldehyde and low concentrations of glutaraldehyde. In the hamster brain, sst2 receptors labelling was observed in both neuronal processes and perikarya. The staining was present in neo-, and allocortical areas of the forebrain, the hypothalamus, brain stem, and spinal cord. In the rat and human, sst1 receptor was shown to be an auto receptor on somatostatinergic neurons located in the hypothalamus. In the retina both sst1 and sst2 receptors were present. sst1 receptors were confined to amacrine cells, few ganglionic cells, and Müller cell-end feet. sst2 receptors were more widespread than the sst1 receptors. sst2-immunoreactivity was present in dopaminergic amacrine cells, the Müller cell-end feet, and in the inner segments of the cone photoreceptors. Thus, the availability of subtype specific antibodies against the five somatostatin receptors makes it possible to identify the receptors involved in the multiple somatostatinergic system in the body.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Photosynthetic electron transfer at the level of cytochrome f and plastocyanin]

Light-induced redox conversions of cytochrome f and plastocyanin in situ and electron transporst from H2O to NADP+ were studied by a dual-wave differential spectrophotometry under identical conditions and subsequently compared. The analysis in red and far red light, treatment by inhibitors, e. g. diurone and dibromothymoquinone, and the analysis of photoreactions during the greening of etiolated seedlings demonstrated that cytochrome f functions only in the non-cyclic chain of electron transport, whereas plastocyanin--both in the non-cyclic and in the cyclic electron transport chains. The jositions of cytochrome f and plastocyanin in various electron-transport chains are proposed.     

Reverse electron transfer from the cytochrome c level in cyanide-resistant mitochondria of the yeast, Candida lipolytica]

The ability of cyanide-resistant mitochondria of yeast Candida lipolytica to perform reverse electron transfer from cytochrome c to alternative oxidase was studied. It was shown that the energy for such a transfer can be provided by high energy intermediates or membrane potential but not by ATP. Reverse electron transfer from cytochrome c is impossible due to energy of NADH and alpha-glycerophosphate oxidation via alternative pathway in the presence of cyanide. These results prove once again that electron transfer via alternative pathway is not connected with the energy accumulation.