Anne-Marie Reboud

Role of acidic phosphoproteins in the partial reconstitution of the active 60 S ribosomal subunit

We have recently shown that rat liver 60 S ribosomal subunits active in protein synthesis can be reconstituted from inactive core particles lacking 30% of the total proteins, mainly L10a, L12, L22, L24, A33 and the acidic phosphoproteins P1–P2, obtained by treatment of 60 S subunits with dimethylmaleic anhydride [(1987) Eur. J. Biochem. 163, 15–20]. In this study, an ethanol extract of the 60 S subunit which contains only P1–P2 was also shown to be effective in reconstitution with the DMMA‐core‐particies: it strongly stimulated the EF‐2‐dependent GTP hydrolysis and, to a lesser extent, polyphenylalanine synthesis; like the DMMA wash it shifted the thermal denaturation curve of the DMMA‐core particles towards that of control subunits. Prior dephosphorylation of the ethanol extract by alkaline phosphatase inhibited the reconstruction process.

Heterogeneity of native rat liver elongation factor 2

The high heterogeneity of native rat liver EF‐2 prepared from either 105000 x g supernatant or microsome high‐salt extract was detected by two‐dimensional equilibrium isoelectric focusing‐SDS‐polyacrylamide gel electrophoresis in the presence of 9.5 M urea. Five spots were always detected, all of M r 95000, which were not artefactual for their amount varied when EF‐2 was specifically ADP‐ribosylated by diphtheria toxin in the presence of NAD+, and/or phosphorylated on a threonine residue by a Ca2+/calmodulin‐dependent protein kinase (most likely Ca2+/calmodulin‐dependent protein kinase III described by others [(1987) J. Biol. Chem. 262, 17299–17303; (1988) Nature 334, 170–173]). Results of ADP‐ribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and [32P]ATP) strongly suggest that our preparation contained native ADP‐ribosylated and native phosphorylated forms which could be estimated at about 20% and 40% of the whole EF‐2. Phosphorylated and ADP‐ribosylated forms of EF‐2 could be ADP‐ribosylated and phosphorylated, respectively, but a native form both ADP‐ribosylated and phosphorylated was not detected. Our results also suggest the existence of a minor native form of EF‐2 and of its phosphorylated and ADP‐ribosylated derivatives.

Modification of the reactivity of three amino-acid residues in elongation factor 2 during its binding to ribosomes and translocation

The accessibility of three amino acids of EF-2, located within highly conserved regions near the N- and C-terminal extremities of the molecule (the E region and the ADPR region, respectively) to modifying enzymes has been compared within nucleotide-complexed EF-2 and ribosomal complexes that mimic the pre- and posttranslocational ones: the high-affinity complex (EF-2)-nonhydrolysable GTP analog GuoPP[CH2]P ribosome and the low-affinity (EF-2)-GDP-ribosome complex, EF-2 and ribosomes being from rat liver. We studied the reactivity of two highly conserved residues diphthamide-715 and Arg-66, to diphtheria-toxin-dependent ADP-ribosylation and trypsin attack, and of a threonine that probably lies between residues 51 and 60, to phosphorylation by a Ca2+/calmodulin-dependent protein kinase. Diphthamide 715 and this threonine residue were unreactive within the high-affinity complex but seemed fully reactive in the low-affinity complex. Arg-66 was resistant to trypsin in both complexes. The possible involvement of the E and ADPR regions of EF-2 in the interaction with ribosome in the two complexes is discussed.

Study on mammalian ribosomal protein reactivity in situ: III. — Effect of trypsin on 40S and 60S subunits

Rat liver 40S and 60S ribosomal subunits were treated with increasing concentrations of trypsin. The activity of both trypsin-treated subunits, when assayed for polyphenylalanine synthesis, progressively decreased, but the 60S subunits were inactivated at much lower trypsin concentrations than were the 40S ones. The sedimentation coefficients of trypsin-treated subunits were identical to those of control subunits when sucrose gradients containing 0.5 M KCl were used. When the sucrose gradients were prepared with a low salt buffer (80 mM KCl), dimer formation was observed with control subunits, but not with trypsin-treated ones. Two-dimensional gel electrophoresis analysis of the proteins extracted from trypsin-treated subunits revealed that all ribosomal proteins in the subunits were accessible to the enzyme. However, several proteins were more resistant to trypsin in compact subunits than when they were free or in unfolded subunits. Proteins of the 60S subunits were generally digested by lower trypsin concentrations than those of the 40S subunits. From the quantitative measurements of the undigested proteins, a classification of the proteins from both subunits according to their trypsin sensitivity was established. These results were compared with those previously obtained concerning ribosomal protein reactivity to chemical reagents.

Action des sels monocationiques sur la dissociation des ribosomes de foie de rat: Proprietes des sous-unites

Abstract Effect of monovalent cations on the dissociation of rat liver ribosomes. Properties of the subunits 1. 1. Rat liver ribosomes, when exposed to concentrated solutions of various monovalent chlorides cations, partly dissociated into subunits. The percentage of subunits depended on the nature of the monovalent cation. Dissociation was enhanced by increasing temperature and salt molarity, or by decreasing Mg2+ and ribosomes concentration. An antagonism was observed between K+ and Li+: small amounts of K+ inhibited the dissociation by Li+. High values of temperature and [monocation] to [Mg2+] ratio led to a partial extraction of ribosomal proteins. 2. 2. In order to prepare subunits, liver ribosomes were centrifuged through a 2 M sucrose layer, containing relatively low concentrations of LiCl, NaCl, KCl, NH4Cl, CsCl (0.3 M) and MgCl2 (0.2 mM) at 4° for 16 h. When the pellets were suspended in a Mg2+ free buffer the dissociation into subunits was complete in every case. Control ribosomes centrifuged without salt did not dissociate. 3. 3. The subunits prepared with KCl and NH4Cl fully reassociated when magnesium was restored. In these conditions, 60–66 % of the subunits obtained with NaCl reassociated. There was almost no reassociation of the subunits prepared with LiCl or CsCl. 4. 4. Ability of control ribosomes and subunits to incorporate [14C]phenylalanine into proteins was examined in the presence and in the absence of polyuridylic acid. The subunits prepared with NaCl, KCl and NH4Cl were respectively 1.3, 1.9 and 1.6 times more active than the control when polyuridylic acid was added. The higher activity of subunits compared with that of ribosomes could not be explained by a shift of the optimal Mg2+ concentration. On the other hand, these subunits had much lower endogenous activities than control ribosomes. Thus washing out the endogenous messenger with salts likely allowed a better binding of polyuridylic acid. The subunits prepared with LiCl and CsCl had a very low activity in every case. 5. 5. The active subunits prepared with salts appeared to be very stable. They could be kept in a Mg2+ free buffer at 4° for several hours without loosing activity. There was no significant decrease of the reassociated subunits activity at 4° after 4 days. In the same conditions control ribosomes lost 22 % of their activity. These results suggest that the method of preparation had removed most of the contaminant nucleases from the subunits. 6. 6. Subunits prepared with salts had a slightly higher density than that of control ribosomes. Therefore they could have lost only a small amount of ribosomal proteins. But, because the RNA extracted from subunits did not appear to be degraded, it remains possible that LiCl and CsCl removed a protein required for biological activity.

Reductive alkylation of mammalian ribosomes.

40- and 60-S ribosomal subunits and 80-S ribosomes from rat liver were highly labelled by reductive methylation using formaldehyde and sodium boro-[3H] hydride, under conditions which did not decrease their activity in poly-U-directed polyphenylalanine synthesis. Dissociation of the monosomes, subunits dimers, and polysomes into free subunits was observed after methylation. Free proteins labelled after extraction from the ribosomal subunits incorporated 7 times more radioactivity than when labelled in the subunits. Proteins extracted from methylated subunits and ribosomes were analyzed by two-dimensional gel electrophoresis, and the radioactivity of each protein was compared to that of the same free protein. A classification of the proteins was established according to their accessibility to the reagents in the subunits and the ribosomes.

Extraction partielle des proteines de ribosomes de foie de rat par differents cations monovalents: Proprietes des particules obtenues

Abstract Partial extraction of proteins from rat liver ribosomes by different monovalent cations. Properties of the core particles 1. Rat liver ribosomes were dissociated into subunits by centrifugation through a sucrose layer containing increasing concentrations of various monovalent cation salts. Poly U-directed polyphenylalanine synthesis by subunits was dependent on the nature and concentration of monovalent cations. Subunits prepared with the following salts: RbCl, KCl, NH4Cl and NaCl had maximum activity when 0.3 M salts were used. The activity of the subunits obtained with 0.3 M RbCl was particularly high. Then activity decreased more or less rapidly according to monovalent cation, when the salt concentration was raised from 0.3 to 0.7 M. The activity of the subunits prepared with other salts (LiCl, CsCl and thallium acetate) continuously decreased with salt concentration without any maximum. When ribosomes were treated with salts at 25° instead of 4°, similar results were obtained, except for NaCl that behaved like the last mentioned salts. 2. Subunits prepared with 0.5–0.7 M monovalent cation salts sedimented in a sucrose gradient a little slower than those obtained with 0.3 M salts or native subunits. In a MgCl2-rich buffer ability of subunits to reassociate varied in the same manner as activity: it was dependent on the monovalent cation used for preparing them, but diminished in every case when salt concentration was raised beyond 0.3 M. 3. Subunits prepared with 0.5 M salts, unlike the one prepared with 0.3 M salts, had a density and a chemical composition markedly different from that of control ribosomes. They had lost between 15 and 36 % of their proteins and then should be considered as incomplete subunits or core particles. The amount of proteins that were extracted depended on the monovalent cation used. The salts could be classified according to their ability to extract ribosomal proteins as follows: NaCl > LiCl > KCl, RbCl > NH4Cl, CsCl. Thallium acetate represented a special case for extracting 27 % proteins even at 0.3 M concentration. 4. Extracted proteins and particle proteins were analysed by polyacrylamide gel electrophoresis. In every case it was observed that salts preferentially extracted some proteins, which were acidic or had relatively high molecular weight. Proteins of subribosomal particles prepared with various salts, when compared together, were found to be not exactly the same. 5. Extracted proteins, when added back to the corresponding core particles, greatly increased the ability of particles to reassociate. The reconstituted material had the same density as control ribosomes. However, activity was not restored when inactive core particles were used for these experiments.

Modifications of 60 S ribosomal subunits induced by the ricin A chain

Incubation of 60 S ribosomal subunits with the ricin A chain reduced their stability during heat treatment. The toxin shifted the thermal denaturation curve of the subunits towards lower temperatures, in a similar way to that produced by the decrease in Mg2+ concentration. A brief heating (3 min at 57°C), which did not affect control subunit activity, enhanced protein synthesis inhibition of the toxin‐treated subunits that released more 5 S RNA, in the form of nucleoprotein complex(es) with protein L5 and phosphoproteins P1P2 (RNPH), than did heated control subunits [(1984) Eur. J. Biochem, 143, 303‐307]. No nuclease activity tested on 60 S subunits and purified 5 S and 5.8 S RNA was found associated with the toxin. The results suggest that the toxin induced a limited conformational change of the 60 S subunit, which destabilized the interaction between RNPH and the rest of the subunit.

Partial in vitro reconstitution of active 40S ribosomal subunits from rat liver

Abstract Treatment of active 40S subunits with high KCl concentrations (1.6 – 2.0 M) resulted in a stepwise extraction of ribosomal proteins. The core particles, which had lost from 27% to 74% of their original proteins, were inactive in protein synthesis. When the missing proteins were added back, active particles, having physical properties similar to those of control 40S were recovered from either 27% or 65% protein-deprived subunits.

Study of mammalian ribosomal protein reactivity in situ. II. - Effect of glutaraldehyde and salts.

Results concerning ribosomal protein sensitivity to glutaraldehyde were compared to protein depletion studies using LiCl centrifugation. The relative degree of reactivity of the different proteins was determined by two-dimensional acrylamide gel electrophoresis, and the activity of the reacted subunits was measured. The results obtained mostly confirmed the studies of methoxynitrotropone reactivity reported earlier. For example, L16, L25, L29, L30, L31, S18, S20 appeared to be definitely exposed to both NH2-reagents and LiCl. Some interesting points emerged from this study regarding protein topography in both subunits: (1) with few exceptions, almost all ribosomal proteins were accessible to the surrounding medium; (2) the sensitivity of the 40S proteins to the three reagents used was lower than was that of the 60S proteins; (3) the reactivities of the subunit components changed when subunits were associated: L8 was more reactive with glutaraldehyde in 60S subunits than in 80S ribosomes. In contrast, S14, S15 and S19 were more exposed in ribosomes than in the 40S subunits.