MITOCHONDRIAL AND CYTOPLASMIC RIBOSOMES FROM TETRAHYMENA PYRIFORMIS : Correlative Analysis by Gel Electrophoresis and Electron Microscopy

Mitochondrial and cytoplasmic ribosomes from Tetrahymena pyriformis have been isolated and studied by the techniques of polyacrylamide gel electrophoresis and electron microscopy used in conjunction. Although the two ribosome types show the same coefficient of sedimentation (80S) in sucrose gradients, they can be distinguished by gel electrophoresis: mitoribosomes migrate in a single band, considerably slower than the cytoribosome band. Electron microscope observations of negatively stained cytoribosomes show typical rounded or triangular profiles, about 275 x 230 Å; mitoribosome profiles are much larger and clearly elongate, about 370 x 240 Å. An electron-opaque spot delimits two nearly equal size subunits. In mixtures of mito- and cytoribosomes, each type can be recognized by its characteristic electrophoretic mobility and by its distinctive fine structure. Cytoribosomal 60S and 40S subunits each produce a distinct electrophoretic band. On the contrary, neither electrophoretic analysis, using a variety of conditions, nor electron microscopy is able to discern two different subunit types in the single 55S mitoribosomal subunit peak. Electrophoretic analysis of RNA shows that both ribosomal RNA species are present in the mitoribosomal subunit fraction. These results establish that mitoribosomes from T. pyriformis dissociate into two subunits endowed with the same sedimentation coefficient, the same electrophoretic mobility, and a similar morphology.     

ELECTRON MICROSCOPIC OBSERVATIONS ON THE LARGE SUBUNIT OF THE RAT LIVER RIBOSOME

Active large subunits obtained by urea treatment of rat liver ribosomes, 59S, were compared with large subunits in intact ribosomes and with the 50S subunits obtained by EDTA treatment. For electron microscopy the specimens were negatively stained or shadow cast. The negatively stained 59S subunits had a slightly ovoidal form; their average dimensions, 244 ± 17 x 207 ± 18 A, were very close to the dimensions of the large subunits in intact ribosomes, and lay between the theoretical dimensions for anhydrous and fully hydrated particles that were calculated from the physical properties of the subunits in solution. The shadow-cast preparations showed particles of similar shape. The 50S subunits, which had lost their 5S RNA, were shadow cast at the same time. They appeared to be more spread out than the 59S subunits and had threadlike extensions. In the positively stained regions of uranyl oxalate-stained preparations the 50S particles varied greatly in shape and size, with average dimensions of 330 ± 21 x 276 ± 33 A, and showed threadlike extensions like those of the shadow-cast particles. For 50S particles in solution the frictional drag of these extensions probably accounts for their low sedimentation coefficient.     

Mitochondrial and cytoplasmic ribosomes. Distinguishing characteristics and a requirement for the homologous ribosomal saltextractable fraction for protein synthesis

1. Mitochondrial and cytoplasmic ribosomes of Euglena gracilis differ in their total RNA and protein content. 2. Mitochondrial ribosomes dissociate to subunits at higher Mg(2+) concentrations than do cytoplasmic ribosomes. 3. A separable 5S RNA is obtained from cytoplasmic and chloroplast ribosomes, but not from mitochondrial ribosomes. 4. For protein-synthesizing activity with a natural mRNA, mitochondrial ribosomes use tRNA from any cell compartment and are partly active with supernatant enzymes from cytoplasm. Cytoplasmic ribosomes are partly active with enzymes and tRNA from mitochondria or chloroplasts. 5. Both mitochondrial and cytoplasmic ribosomes show high specificity for the homologous salt-extractable ribosomal fraction for protein-synthesizing activity.     

LOW RESISTANCE JUNCTIONS IN CRAYFISH : II. Structural Details and Further Evidence for Intercellular Channels by Freeze-Fracture and Negative Staining

Low resistance junctions between axons of crayfish ganglia are studied by freeze-fracture and negative staining. In freeze-fracture, fracture planes that go through a junctional membrane expose two faces, both internal, called face A and face B. Face A belongs to the internal membrane leaflet and faces the gap. Face B belongs to the external membrane leaflet and faces the axoplasm. Face A displays pits, 60–100 Å in diameter, arranged in a hexagonal array with a unit cell of ~200 Å. An ~25 Å bump is frequently seen at the center of each pit. Some pits are occupied by a globule ~125 Å in diameter, which displays a central depression ~25 Å in size. Face B contains globules also arranged in a fairly regular hexagonal pattern. The center-to-center distance between adjacent globules is most frequently ~200 Å; however, occasionally certain globules are seen separated by a distance as short as ~125 Å. The top surface of the globules occasionally displays a starlike profile and seems to contain a central depression ~25 Å in diameter. In negatively stained preparations of membranes from the nerve cord, two types of membranes are seen containing a fairly regular pattern. In one, globules ~95 Å in diameter form a hexagonal close packing with a unit cell of ~95 Å. In the other, globules of the same size are organized in a larger hexagonal array with a unit cell of ~155 Å (swollen arrangement). Some of the globules forming the swollen arrangement are seen containing six subunits. The six subunits form a hexagon which is skewed with respect to the main rows of hexagons in such a way that the subunits lie on rows which make an angle of ~37° with the main rows.     

Mitochondrial and cytoplasmic ribosomes from mammalian tissues. Further characterization of ribosomal subunits and validity of buoyant-density methods for determination of the chemical composition and partial specific volume of ribonucleoprotein particles

1. At 0-4 degrees C mitochondrial ribosomes (55S) dissociate into 39S and 29S subunits after exposure to 300mm-K(+) in the presence of 3.0mm-Mg(2+). When these subunits are placed in a medium containing a lower concentration of K(+) ions (25mm), approx. 75% of the subparticles recombine giving 55S monomers. 2. After negative staining the large subunits (20.3nm width) usually show a roundish profile, whereas the small subunits (12nm width) show an elongated, often bipartite, profile. The dimensions of the 55S ribosomes are 25.5nmx20.0nmx21.0nm, indicating a volume ratio of mitochondrial to cytosol ribosomes of 1:1.5. 3. The 39S and 29S subunits obtained in high-salt media at 0-4 degrees C have a buoyant density of 1.45g/cm(3); from the rRNA content calculated from buoyant density and from the rRNA molecular weights it is confirmed that the two subparticles have weights of 2.0x10(6) daltons and 1.20x10(6) daltons; the weights of the two subunits of cytosol ribosomes are 2.67x10(6) and 1.30x10(6) daltons. 4. The validity of the isodensity-equilibrium-centrifugation methods used to calculate the chemical composition of ribosomes was reinvestigated; it is confirmed that (a) reaction of ribosomal subunits with 6.0% (v/v) formaldehyde at 0 degrees C is sufficient to fix the particles, so that they remain essentially stable after exposure to dodecyl sulphate or centrifugation in CsCl, and (b) the partial specific volume of ribosomal subunits is a simple additive function of the partial specific volumes of RNA and protein. The RNA content is linearly related to buoyant density by the equation RNA (% by wt.)=349.5-(471.2x1/rho(CsCl)), where 1/rho(CsCl)=[unk](RNP) (partial specific volume of ribonucleoprotein). 5. The nucleotide compositions of the two subunit rRNA species of mitochondrial ribosomes from rodents (42% and 43% G+C) are distinctly different from those of cytoplasmic ribosomes.     

Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits

Highly purified mitochondrial ribosomes (mitoribosomes) have been obtained from the yeast Candida utilis. Sedimentation analysis in sucrose gradients made in 5 mM MgCl₂, 1 mM Tris, pH 7.4 and 50 mM KCl clearly distinguishes mitoribosomes (72S) from cytoplasmic ribosomes (cytoribosomes) (78S). Mitoribosomes are completely dissociated into 50S and 36S subunits at 10^-4 M MgCl₂ whereas complete dissociation of cytoribosomes into 61S and 37S subunits occurs only at 10^-6 M MgCl₂ Electron microscopy of negatively stained mitoribosomes (72S peak) shows bipartite profiles, about 265 x 210 x 200 A Characteristic views are interpreted as frontal, dorsal, and lateral projections of the particles, the latter is observed in two enantiomorphic forms Mitoribosome 50S subunits display rounded profiles bearing a conspicuous knoblike projection, reminiscent of the large bacterial subunit. The 36S subunits show a variety of angular profiles. Mitoribosomal subunits are subject to artifactual dimerization at high Mg²⁺ concentration Under these conditions, a supplementary 80S peak arises. Electron microscopic observation of the 80S peak reveals closely paired particles of the 50S type Buoyant density determinations after glutaraldehyde fixation show a single peak at ρ = 1.48 for mitoribosomes and 1.53 for cytoribosomes In the presence of ethylenediaminetetraacetate (EDTA), two species of RNA, 21S and 16S, are obtained from mitoribosomes, while 25S and 17S RNA are obtained from cytoribosomes It is established that the small and large RNA species are derived from the 36S and 50S subunits, respectively, by extraction of the RNA from each subunit The G + C content of the RNA is lower for mitoribosomes (33%) than for cytoribosomes (50%). Incubation of C utilis mitochondria with leucine-¹⁴C results in the labeling of 72S mitoribosomes. The leucine-¹⁴C incorporation is inhibited by chloramphenicol and resistant to cycloheximide Puromycin strips the incorporated radioactivity from the 72S mitoribosomes, which is consistent with the view that leucine-¹⁴C is incorporated into nascent polypeptide chains at the level of mitoribosomes     

Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA

So far, four RNA:pseudouridine (Ψ)-synthases have been identified in yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Ψ-synthases responsible for several U→Ψ conversions in tRNAs and UsnRNAs remained to be identified. Based on conserved amino-acid motifs in already characterised RNA:Ψ-synthases, four additional open reading frames (ORFs) encoding putative RNA:Ψ-synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Ψ residue normally present in the fully matured mitochondrial rRNAs (Ψ₂₈₁₉ in 21S rRNA) was missing, while Ψ residues at all the tested pseudouridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U→Ψ conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71→A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Ψ₂₈₁₉ synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Ψ-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Ψ₂₈₁₉ in mitochondrial 21S rRNA is not essential for cell viability.     

LOW RESISTANCE JUNCTIONS IN CRAYFISH : I. Two Arrays of Globules in Junctional Membranes

The ultrastructure of low resistance junctions between segments of lateral giant fibers in crayfish is studied in sections from specimens fixed either by conventional methods or by glutaraldehyde-H₂O₂ or by glutaraldehyde-lanthanum. Cross sections through junctions fixed by conventional glutaraldehyde display the usual trilaminar profile of two parallel membranes separated by a narrow gap. Most of the junctional regions appear covered by 500–800 Å vesicles which lie on both sides of the junction in rows adjacent to the membranes. Gross sections through junctions fixed by glutaraldehyde-H₂O₂ display, in regions containing vesicles, membranes with a beaded profile. The beads correspond to globules ~125 Å in width and ~170 Å in height arranged in a hexagonal pattern with a unit cell of ~200 Å. The globules of one membrane match precisely with those of the adjacent membrane, and opposite globules seem to come in contact with each other at the center of the junction. The membrane of the vesicles also contains globules. Occasionally the globules of the vesicles seem to join with those of the junctional membranes, apparently forming intracellular junctions. Injunctions negatively stained by lanthanum the globules are seen organized into two arrangements. Areas containing globules in a hexagonal array with a unit cell of ~200 Å (swollen pattern) are seen adjacent to areas in which the globules are more closely and disorderly packed (close packing), the minimum center-to-center distance between adjacent globules being ~125 Å. At higher magnification each globule appears composed of six subunits arranged in a circle around a central region occupied by lanthanum (possibly a pit).     

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.     

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem–loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K_M for substrate 70S ribosomes is 0.51 ± 0.06 μM, and for cofactor S-adenosyl-L-methionine 27 ± 3 μM; the k_cat values are 4.95 ± 1.10 min⁻¹ and 6.4 ± 1.3 min⁻¹, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K_M value for substrate 50S subunits is 0.98 ± 0.18 μM and the k_cat value is 1.97 ± 0.46 min⁻¹. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD- and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.     

Crystallization of 70 S ribosomes and 30 S ribosomal subunits from Thermus thermophilus

Well-ordered three-dimensional crystals of 70 S ribosomes and 30 S ribosomal subunits from extremely thermophilic bacteria Thermus thermophilus have been obtained. Positively stained thin sections of the crystals have been analyzed by electron microscopy. Redissolved crystalline ribosomes and small ribosomal subunits reveal sedimentation constants of 70 S and 30 S, respectively, and are functionally active in the poly(U)-system.     

SUBSTRUCTURE OF AMPHIBIAN MOTOR END PLATE : Evidence for a Granular Component Projecting from the Outer Surface of the Receptive Membrane

End-plate membrane has been examined at amphibian myoneural junctions by means of transmission electron microscopy of thin tissue sections. The postjunctional membrane exhibits morphologically specialized dense, convex patches which are located superficially facing the axon terminal but do not extend into the depths of the junctional folds. In the specialized regions the plasma membrane is ~ 120 Å thick and trilaminar. The outer dense lamina is thickened by the presence in it of granular elements ~60–120 Å in diameter which are spaced semiregularly at ~100–150-Å intervals and which border the junctional cleft directly. In these regions the concentration of the granules is of the order of ~ 10⁴/µm², which is in the same range as the estimated concentration of receptor sites at other vertebrate cholinergic junctions. Filamentous projections can sometimes be seen extending from the granules to the overlying basement membrane, and in oblique views a reticular pattern may appear both in these patches and in the basement membrane. The cytoplasmic surface of the specialized membrane is covered with an amorphous and filamentous dense material whose distribution coincides with that of the granules visible in the outer layer and which may be connected to them across the membrane. In unosmicated specimens stained with permanganate and uranyl acetate the specialized regions exhibit the same morphological features but stand out sharply in contrast to adjacent areas of unspecialized membrane which appear only faintly. Such preparations are particularly useful in assessing the extent of the specialized membrane. It is proposed that the granules visible at the outer surface of the end-plate membrane represent acetylcholine receptors and that in amphibians, as in annelids, the receptors at myoneural junctions are concentrated into patches which occupy less than the total postjunctional membrane surface area.     

Nucleo-cytoplasmic interactions in the petite negative yeast Schizosaccharomyces pombe

Summary In the petite positive yeast, Saccharomyces cerevisiae, cycloheximide selectively inhibits protein synthesis on cytoplasmic ribosomes, and, as a consequence, nuclear DNA synthesis. Mitochondrial DNA, however, is synthesized for 4–6 h after cessation of protein synthesis. In this paper we show that in contrast to Saccharomyces cerevisiae, synthesis of mitochondrial and nuclear DNA is tightly coordinated in the petite negative yeast Schizosaccharomyces pombe, since inhibition of cytoplasmic protein synthesis leads immediately to cessation of both nuclear and mitochondrial DNA synthesis.Dedicated to Prof. Dr. F. Kaudewitz on occasion of his 60th birthday     

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D₃₇ values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 × 10⁵ and 1.4 × 10⁵, were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 × 10⁵) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.     

A sensitive in vitro protein synthesizing system from Ehrlich ascites mitochondria

The S-25 fraction prepared from digitonin washed mitochondria is highly active in poly(U) directed phenylanine incorporation when supplemented with t-RNA. Ribosomes prepared from the S-25 fraction contain 58S monomeric ribosomes and 40S and 29S subunits. Further, these ribosomes contain 21S and 13S rRNA. No detectable cytoplasmic specific ribosomal particles and also rRNA was detected in the mitochondrial S-25 preparation. Ribosomes from mitochondrial S-25 have specific requirement for mitochondrial specific supernatant factors for complete activity.     

TELOPHASE SEGREGATION OF CHROMOSOMES AND AMITOSIS

Markedly improved fixation of leaf tissues is obtained by means of a glutaraldehyde (or acrolein)-osmium tetroxide procedure, as compared with the results of potassium permanganate or osmium tetroxide fixation methods. The procedure has proved useful in all species so far examined. Chloroplasts are particularly well preserved. In this paper details of components of the ground-substance of Avena sativa plastids are presented. They include the following:—(i) The "tromacentre" is an area of aggregated fibrils, each 85 A in diameter, and of uncertain length. Individual fibrils may be composed of subunits. The whole aggregate is usually up to 1 µ in diameter, and is visible in thin sections in the light microcope. It is present at all stages of plastid development, and, under conditions of rapid synthesis in the plastid, it may be up to 2 µ in diameter. Evidence that it is proteinaceous is presented. Osmiophilic globules are often associated with it. (ii) Areas which resemble bacterial and blue-green algal nucleoplasms, containing fibrils approximately 30 A wide. These regions are smaller than the stromacentre and, like that structure, they occur in all stages of plastid development. Unlike it, there are several such areas per chloroplast. (iii) Particles which have some of the morphological and staining characteristics of ribosomes. Present at all stages of development, they are approximately two-thirds the size of the cytoplasmic ribosomes. They can occur in groups, thus resembling polyribosomes. (iv) The remaining material is granular, and may include dissociated portions of stromacentre material. The validity of the observations and their significance is discussed.     

RABBIT SKELETAL MUSCLE GLYCOGEN : A Morphological and Biochemical Study of Glycogen β-Particles Isolated by the Precipitation-Centrifugation Method

Glycogen in its particulate β-form is localized in the sarcoplasm close to the sarcoplasmic reticulum. Some particles are in close contact with the membranes, on the outer side of the vesicles. The mild technique of differential precipitation-centrifugation has been adapted to the preparation of glycogen from adult skeletal muscle. A preliminary low-speed centrifugation which eliminates the contractile protein structures and the cell debris is followed by a high-speed centrifugation which produces pellets containing glycogen mixed with smooth-walled vesicles, the glycogen-sarcovesicular fraction. The glycogen obtained after treatment of this fraction with deoxycholate and two washings contains 3% protein. A similar protein content contaminates glycogen banded in a linear sucrose gradient. The glycogen-sarcovesicular fraction and the purified glycogen have been examined, under the electron microscope, in sections of fixed and embedded material or with the negative staining technique. The glycogen β-particles in negatively stained preparations have an average diameter of 39.4 mµ. The largest particles present irregular outlines, suggesting the presence of conglomerated subunits, about 20 mµ in diameter. These subunits seem to fall apart under the influence of concentrated potassium hydroxide. The mean sedimentation coefficients calculated for infinite dilution vary from 115 to 135S. The spectrophotometric analysis of the glycogen-iodine complex indicates the presence of long end-chains in the molecule.     

The alpha-subunit of the maize F(1)-ATPase is synthesised in the mitochondrion

The F₁-ATPase complex has been purified from maize (Zea mays L.) mitochondria and shown to consist of five subunits with mol. wts. of 58 000 (α), 56 000 (β), 35 000 (γ), 22 000 (δ) and 8000 (ε). The α-subunit co-migrates on one- and two- dimensional isoelectric focussing-SDS polyacrylamide gels with the major polypeptide synthesised by isolated mitochondria. One-dimensional proteolytic peptide mapping and immunoprecipitation confirms that the α-subunit is a mitochondrial translation product and therefore presumably encoded in mitochondrial DNA. This contrasts with the situation in animal and fungal cells where all five subunits of the F₁-ATPase are encoded by the nuclear genome and synthesised on cytosolic ribosomes.     

IMMUNOHISTOCHEMICAL LOCALIZATION OF CONTRACTILE PROTEINS IN LIMULUS STRIATED MUSCLE

Limulus paramyosin and myosin were localized in the A bands of glycerinated Limulus striated muscle by the indirect horseradish peroxidase-labeled antibody and direct and indirect fluorescent antibody techniques. Localization of each protein in the A band varied with sarcomere length. Antiparamyosin was bound at the lateral margins of the A bands in long (~ 10.0 µ) and intermediate (~ 7.0 µ) length sarcomeres, and also in a thin line in the central A bands of sarcomeres, 7.0–~6.0 µ. Antiparamyosin stained the entire A bands of short sarcomeres (<6.0). Conversely, antimyosin stained the entire A bands of long sarcomeres, showed decreased intensity of central A band staining except for a thin medial line in intermediate length sarcomeres, and was bound only in the lateral A bands of short sarcomeres. These results are consistent with a model in which paramyosin comprises the core of the thick filament and myosin forms a cortex. Differential staining observed using antiparamyosin and antimyosin at various sarcomere lengths and changes in A band lengths reflect the extent of thick-thin filament interaction and conformational change in the thick filament during sarcomeric shortening.     

Reduced Dosage of Genes Encoding Ribosomal Protein S18 Suppresses a Mitochondrial Initiation Codon Mutation in Saccharomyces Cerevisiae

A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18aΔ and rps18bΔ null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18aΔ then rps18bΔ. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products.