Ada Zamir

Inactivation and reactivation of ribosomal subunits: Amino acyl-transfer RNA binding activity of the 30 s subunit of Escherichia coli☆

Abstract The ability of the 30 s ribosomal subunit to bind phenylalanyl-transfer RNA in the cold in response to polyuridylic acid is lost if the subunit is subjected, even transiently, to either of two treatments: (a) the removal of certain specific monovalent cations (NH+4, K+, Rb+ or Cs+), or (b) the reduction of the Mg2+ concentration below a critical concentration of about 2 m m . If the depleted cation is restored, the subunit reverts to an active form in a process that is greatly enhanced by heat. Thermally reactivated subunits retain full activity when rechilled, showing that the inactivation and reactivation processes involve changes, presumably conformational, in the subunit itself. Reactivation follows first-order kinetics with respect to the appearance of active subunits, with an Arrhenius activation energy of 26 kcal./mole between 30 °C and 40 °C. On storage at 0 °C, inactive 30 s subunits gradually lose the ability to be reactivated. Part of this loss is due to the oxidation of one or more sulphydryl groups and is prevented or reversed if a sulphydryl reducing agent is included in the storage or the reactivation medium, respectively. Active and inactive 30 s subunits have the same sedimentation coefficient and there is no direct evidence that they differ in conformation. However, two kinds of indirect evidence are in accord with the existence of conformational differences: (a) under appropriate conditions inactive 30 s subunits form dimers sedimenting at 50 s while active subunits do not, and active 30 s subunits associate more readily with 50 s subunits to form 70 s ribosomes; (b) inactive 30 s subunits undergo sulphydryl oxidation much more rapidly than do active ones. Although differing in certain details, the 30 s inactivation and reactivation processes are generally similar to those previously described for the 50 s subunit. Both subunits can exist in active and inactive forms which are easily and reversibly interconverted, suggesting that the structure of the functional ribosome is flexible and easily altered. The interconversions affect a number of ribosomal activities in parallel. It is possible that many previously described phenomena pertaining to ribosomal activity can be interpreted, at least in part, in terms of ribosomal inactivation and reactivation.

A Salt-resistant Plasma Membrane Carbonic Anhydrase Is Induced by Salt in Dunaliella salina

The mechanisms allowing proliferation of the unicellular green alga Dunaliella salina in up to saturating NaCl concentrations are only partially understood at present. Previously, the level of a plasma membrane Mr 60,000 protein, p60, was found to increase with rising external salinities. Based on cDNA cloning and enzymatic assays, it is now shown that p60 is an internally duplicated carbonic anhydrase, with each repeat homologous to animal and Chlamydomonas reinhardtii carbonic anhydrases, but exceptional in the excess of acidic over basic residues. Increasing salinities, alkaline shift, or removal of bicarbonate induced in D. salina parallel increases in the levels of p60, its mRNA, and external carbonic anhydrase activity. Moreover, purified p60 exhibited carbonic anhydrase activity comparable to other carbonic anhydrases. A p60-enriched soluble preparation showed maximal carbonic anhydrase activity at ∼1.0 M NaCl and retained considerable activity at higher salt concentrations. In contrast, a similar preparation from C. reinhardtii was ∼90% inhibited in 0.6 M NaCl. These results identified p60 as a structurally novel carbonic anhydrase transcriptionally regulated by CO2 availability and exhibiting halophilic-like characteristics. This enzyme is potentially suited to optimize CO2 uptake by cells growing in hypersaline media.

Inactivation and reactivation of ribosomal subunits: The peptidyl transferase activity of the 50 s subunit of Escherichia coli

The ability of the 50 s ribosomal subunit to catalyze the peptidyl transferase reaction is absolutely dependent on the continued presence of certain monovalent cations in the ribosomal medium. This ability is maintained as long as one of these cations is present and is lost if the cation is removed. The effective cations are NH4+ ≧ Rb+ > K+ > Cs+. Na+ and Li+ are ineffective. Activity is restored, with the same order of effectiveness, if an appropriate cation is added back. Although at high salt concentrations there may be a slow reactivation in the cold, heat greatly accelerates the process at all salt concentrations. Although reactivating cations are present in the peptidyl transferase assay medium, inactive ribosomes remain inactive if the assay conditions do not promote reactivation; i.e. if the assay is performed in the cold. This shows that the effect is on the ribosome and not on the assay. The ribosome can exist in two different states, active and inactive, and can be brought from either state to the other. The rate of reactivation increases markedly with rising temperature or monovalent cation concentration. Reactivation is also accelerated by the presence of aliphatic alcohols in the medium and by substrate, fMet-tRNA, provided that the substrate is actually bound to the ribosome during the heat treatment. Reactivation of ribosomes follows first-order kinetics under a wide variety of conditions. The energy of activation of the process is 40 to 50 kcal./mole below 30 °C but becomes less at higher temperatures. Inactive subunits lack no essential components and have the same sedimentation pattern and coefficient as active ones. However, it seems likely that the interconversion between the active and inactive form involves a conformational change. It is not known if the inactivation-activation process occurs in vivo; the possibility is discussed.

Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina.

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant β-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 m NaCl, as did the level of thekcs mRNA. The function of the kcsgene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing thekcs cDNA. The effect of salinity on kcsexpression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 mNaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5m salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.

A Structurally Novel Transferrin-like Protein Accumulates in the Plasma Membrane of the Unicellular Green Alga Dunaliella salina Grown in High Salinities

The alga Dunaliella salina is outstanding is its ability to withstand extremely high salinities. To uncover mechanisms underlying salt tolerance, a search was carried out for salt-induced proteins. The level of a plasma membrane 150-kDa protein, p150, was found to increase with rising external salinity (Sadka, A., Himmelhoch, S., and Zamir, A. (1991) Plant Physiol. 95, 822-831). Based on its cDNA-deduced sequence, p150 belongs to the transferrin family of proteins so far identified only in animals. This, to our best knowledge, is the first demonstration of a transferrin-like protein in a photosynthetic organism. Unlike animal transferrins, p150 contains three, rather than two, internal repeats and a COOH-terminal extension including an acidic amino acid cluster. In intact cells p150 is degraded by Pronase, indicating that the protein is extracellularly exposed. The relationship of p150 to iron uptake is supported by the induction of the protein in iron-deficient media and by its radioactive labeling in cells grown with 59Fe. Accumulation of p150 is transcriptionally regulated. It is proposed that p150 acts in iron uptake other than by receptor-mediated endocytosis and that its induction permits the cells to overcome a possible limitation in iron availability under high salinities.

[40] The inactivation and reactivation of Escherichia coli ribosomes

Publisher Summary When E. coli ribosomes or either of their subunits are assayed under certain conditions for any of a number of biological activities, they can be shown to exist in one of several different states: active, inactive, or partially active. The state is determined by the past treatment of the ribosome. These states are reversibly inter convertible and ribosomes can be brought from one to another by relatively mild treatments. Among the factors that influence these inter conversions in vitro are temperature, the ionic environment, and interactions between subunits or between the ribosome and certain other macromolecules that participate in protein synthesis. 70 S, 50 S, and 30 S ribosomes all undergo these inter conversions, although the characteristics of the process may vary somewhat in each case.

N-ethyl maleimide as a probe for the study of functional sites and conformations of 30 S ribosomal subunits

Escherichia coli 30 S ribosomal subunits are inactive in a number of specific functions when Mg2+ concentration is reduced to 1 mM, and activity is recovered on heating under appropriate ionic conditions. When active and inactive forms were treated with N-ethyl maleimide, both forms reacted to a similar extent, but the reagent attached mostly to different proteins. Moreover, it caused irreversible inactivation only when reacting with the inactive form of the subunit. Though the activating treatment failed to restore activity to these subunits it did expose the same sulfhydryl groups as are available in the active state for reaction with the maleimide. Different ribosomal activities were eliminated at different maleimide concentrations, permitting the assignment of specific functions to sulfhydryl groups of specific ribosomal proteins. Protein S18 appears to be involved in subunit association, binding of fMet-tRNA and of aminoacyl-tRNA to the P-site. Proteins S1, S14 and S21 are all or in part involved in the binding of aminoacyl-tRNA to the A-site and in the binding of the antibiotic dihydrostreptomycin. The reaction with N-ethyl maleimide thus provides a criterion other than biological activity for characterizing different ribosomal forms and a tool for mapping the 30 S subunit for specific functional sites.

Iron Uptake by the Halotolerant Alga Dunaliella Is Mediated by a Plasma Membrane Transferrin

A 150-kDa transferrin-like protein (Ttf) is associated with the plasma membrane of the halotolerant unicellular alga Dunaliella salina (Fisher, M., Gokhman, I., Pick, U., and Zamir, A. (1997) J. Biol. Chem. 272, 1565–1570). The Ttf level rises with medium salinity or upon iron depletion. Evidence that Ttf is involved in iron uptake by Dunaliellais presented here. Algal iron uptake exhibits characteristics resembling those of animal transferrins: high specificity and affinity for Fe3+ ions, strict dependence on carbonate/bicarbonate ions, and very low activity in acidic pH. Reducing the level of Ttf by mild proteolysis of whole cells is accompanied by lowered uptake activity. Conversely, accumulation of high levels of Ttf is correlated with an enhancement of iron uptake. Kinetically, iron uptake consists of two steps: an energy-independent binding of iron to the cell surface and an energy-dependent internalization. Salinities as high as 3.5 m NaCl do not inhibit iron uptake or decrease the apparent affinity for Fe3+ ions, implying that Ttf activity is not affected by high salt. These results indicate that transferrins, hitherto identified only in animals, are present and function in iron transport also in plant systems.

Interconversions between inactive and active forms of ribosomal subunits

We have previously reported that the peptidyl transferase activity of the 50 S subunit of E. coli ribosomes is lost if the ribosomes are exposed to media lacking NH: and K’. Activity can be restored, but this requires both (a) the readdition of one of these ions, and (b) heat. The heat requirement is discerned only if the ribosomes are assayed at O’, where they are active only if they were previously heated in the presence of NH: or K+ [ 11. We have now studied a specific function of the 30 S subunit, the non-enzymatic binding of phenylalanyltRNA directed by poly U and assayed at 0”, and have found a similar inactivation and reactivation, with similar effects of specific monovalent cations and heat. In addition, the 30 S subunit is inactivated if the M

A Salt-Induced 60-Kilodalton Plasma Membrane Protein Plays a Potential Role in the Extreme Halotolerance of the Alga Dunaliella

+ concentration is lowered to 1 mM (as is commonly done to dissociate 70 S ribosomes), even if NH: is present. It is of particular interest, however, that ribosomes that have been inactivated toward non-enzymatic binding are active at 0’ in the enzymatic binding reactions mediated by initiation factors or transfer factor T, even if they have not been previously heated.