Assembly Of Large Subunits Research Articles

Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli.

Hydrogenases are abundant metalloenzymes that catalyze the reversible conversion of molecular H2 into protons and electrons. Important achievements have been made over the past two decades in the understanding of these highly complex enzymes. However, most hydrogenases have low production yields requiring many efforts and high costs for cultivation limiting their investigation. Heterologous production of these hydrogenases in a robust and genetically tractable expression host is an attractive strategy to make these enzymes more accessible. In the present study, we chose the oxygen-tolerant H2-sensing regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha H16 owing to its relatively simple architecture compared to other [NiFe]-hydrogenases as a model to develop a heterologous hydrogenase production system in Escherichia coli. Using screening experiments in 24 deep-well plates with 3 mL working volume, we investigated relevant cultivation parameters, including inducer concentration, expression temperature, and expression time. The RH yield could be increased from 14 mg/L up to >250 mg/L by switching from a batch to an EnPresso B-based fed-batch like cultivation in shake flasks. This yield exceeds the amount of RH purified from the homologous host R. eutropha by several 100-fold. Additionally, we report the successful overproduction of the RH single subunits HoxB and HoxC, suitable for biochemical and spectroscopic investigations. Even though both RH and HoxC proteins were isolated in an inactive, cofactor free apo-form, the proposed strategy may powerfully accelerate bioprocess development and structural studies for both basic research and applied studies. These results are discussed in the context of the regulation mechanisms governing the assembly of large and small hydrogenase subunits.

Role of GTPases in Driving Mitoribosome Assembly.

Mitoribosomes catalyze essential protein synthesis within mitochondria. Mitoribosome biogenesis is assisted by an increasing number of assembly factors, among which guanosine triphosphate hydrolases (GTPases) are the most abundant class. Here, we review recent progress in our understanding of mitoribosome assembly GTPases. We describe their shared and specific features and mechanisms of action, compare them with their bacterial counterparts, and discuss their possible roles in the assembly of small or large mitoribosomal subunits and the formation of the monosome by establishing quality-control checkpoints during these processes. Furthermore, following the recent unification of the nomenclature for the mitoribosomal proteins, we also propose a unified nomenclature for mitoribosome assembly GTPases.

BSD2 is a Rubisco-specific assembly chaperone, forms intermediary hetero-oligomeric complexes, and is nonlimiting to growth in tobacco.

The folding and assembly of Rubisco large and small subunits into L8 S8 holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco-BSD2 assembly complexes in the model C3 plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70nmol BSD2 protomer.m2 ) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L8 S8 pool. RNAi-silencing BSD2 production in transplastomic tobacco producing bacterial L2 Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi-bsd2 alleles into wild-type tobacco however impaired L8 S8 Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2-silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.

Ribosomal Proteins Control or Bypass p53 during Nucleolar Stress.

The nucleolus is the site of ribosome biogenesis, a complex process that requires the coordinate activity of all three RNA polymerases and hundreds of non-ribosomal factors that participate in the maturation of ribosomal RNA (rRNA) and assembly of small and large subunits. Nevertheless, emerging studies have highlighted the fundamental role of the nucleolus in sensing a variety of cellular stress stimuli that target ribosome biogenesis. This condition is known as nucleolar stress and triggers several response pathways to maintain cell homeostasis, either p53-dependent or p53-independent. The mouse double minute (MDM2)-p53 stress signaling pathways are activated by multiple signals and are among the most important regulators of cellular homeostasis. In this review, we will focus on the role of ribosomal proteins in p53-dependent and p53-independent response to nucleolar stress considering novel identified regulators of these pathways. We describe, in particular, the role of ribosomal protein uL3 (rpL3) in p53-independent nucleolar stress signaling pathways.

HALP, A Novel Transcriptional U Three Protein (t-UTP), Activates RNA Polymerase I Transcription by Binding and Acetylating the Upstream Binding Factor (UBF)

Transcription of ribosome RNA precursor (pre-rRNA) and pre-rRNA processing are coordinated by a subset of U three proteins (UTPs) known as transcriptional UTPs (t-UTPs), which participate in pre-rRNA transcription in addition to participation in 18 S rRNA processing. However, the mechanism by which t-UTPs function in pre-rRNA transcription remains undetermined. In the present study, we identified hALP, a histone acetyl-transferase as a novel t-UTP. We first showed that hALP is nucleolar, and is associated with U3 snoRNA and required for 18 S rRNA processing. Moreover, depletion of hALP resulted in a decreased level of 47 S pre-rRNA. Ectopic expression of hALP activated the rDNA promoter luciferase reporter and knockdown of hALP inhibited the reporter. In addition, hALP bound rDNA. Taken together these data identify hALP as a novel t-UTP. Immunoprecipitation and GST pulldown experiments showed that hALP binds the upstream binding factor (UBF) in vivo and in vitro. It is of importance that hALP acetylated UBF depending on HAT in vivo, and hALP but not hALP (ΔHAT) facilitated the nuclear translocation of the RNA polymerase I (Pol I)-associated factor 53 (PAF53) from the cytoplasm and promoted the association of UBF with PAF53. Thus, we provide a mechanism in which a novel t-UTP activates Pol I transcription by binding and acetylating UBF.

Phosphorylation of rubisco in Cicer rietinum: Non-phosphoprotein nature of rubisco in Nicotiana tabacun

Leaf discs from Cicer and Nicotiana were incubated in a solution of [ 32P]-orthophosphate (24 hr). RUBISCO was purified from these and electrophoresed on native PAGE. A single protein stained band was observed on gels, thus proving the electrophoretic homogeneity of RUBISCO both in Cicer and Nicotiana. Autoradiography of gels revealed a radioactive band in the zone of protein stained band of Cicer RUBISCO, whereas no radioactivity was detected in the region of the protein stained band of Nicotiana RUBISCO. This suggested that Cicer RUBISCO unlike that of Nicotiana is a phosphoprotein. Further analysis of [ 32P]-labelled purified Cicer RUBISCO on SDS—PAGE showed two protein stained bands corresponding to the large and small subunits of the enzyme. Autoradiography of the denaturing gel showed a radioactive band exclusively in the zone of the small subunit. Chemical characterization of [ 32P]-labelled Cicer RUBISCO by acid hydrolysis showed radioactivity in phosphoserine and phosphotyrosine. Treatment of [ 32P]-labelled Cicer RUBISCO with purified alkaline phosphatase brought about a significant dephosphorylation (∼70%) of the enzyme. The in vitro dephosphorylation of Cicer RUBISCO resulted in the dissociation of the small subunits from the catalytic octameric large subunits. Concomitantly, there was a marked decrease in the catalytic activity of the dephosphorylated Cicer RUBISCO in comparison with the phosphorylated form of RUBISCO. Phosphorylation of the small subunit seems crucial for the assembly of large and small subunits and thus contributes to the regulation of Cicer RUBISCO. By contrast, Nicotiana RUBISCO showed no loss of catalytic activity following its treatment with alkaline phosphatase.

Synthesis and Assembly of Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase in Chloroplast Extracts

We have developed a new system for the in vitro synthesis of large subunits and their assembly into ribulose bisphosphate carboxylase oxygenase (Rubisco) holoenzyme in extracts of higher plant chloroplasts. This differs from previously described Rubisco assembly systems because the translation of the large subunits occurs in chloroplast extracts as opposed to isolated intact chloroplasts, and the subsequent assembly of large subunits into holoenzyme is completely dependent upon added small subunits. Amino acid incorporation in this system displayed the characteristics previously reported for chloroplast-based translation systems. Incorporation was sensitive to chloramphenicol or RNase but resistant to cycloheximide, required magnesium, and was stimulated by nucleotides. The primary product of this system was the large subunit of Rubisco. However, several lower molecular weight polypeptides were formed. These were structurally related to the Rubisco large subunit. The initiation inhibitor aurintricarboxylic acid (ATA) decreased the amount of lower molecular weight products accumulated. The accumulation of completed large subunits was only marginally reduced in the presence of ATA. The incorporation of newly synthesized large subunits into Rubisco holoenzyme occurred under conditions previously identified as optimal for the assembly of in organello-synthesized large subunits and required the addition of purified small subunits.

Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.

Mitochondrial gene expression in a nuclear mutant of Neurospora deficient in large subunits of mitochondrial ribosomes

A new cytochrome a and b deficient nuclear mutant of Neurospora crassa, cyt-U-28, is defective in the assembly of large subunits of mitochondrial ribosomes. Nonetheless, this mutant overproduces apparently normal small subunits of mitochondrial ribosomes, even though it should be deficient for the S5 ribosomal protein required for assembly of the particles beyond the CAP30S stage. The mitochondria of cyt-U-28 indeed synthesize only small amounts of most mitochondrial polypeptides, including cytochrome oxidase subunits I, II, and III, contain very low amounts of the normal seven-polypeptide cytochrome oxidase complex, and, unlike the organelles from other cytochrome a deficient mutants, do not accumulate the nuclear-encoded cytochrome oxidase subunits 5 and 6. Nonetheless, the mutant markedly oversynthesizes a mitochondrial protein that comigrates with subunit 9 of the mitochondrial ATPase on S DS–polyacrylamide electrophoresis gels. The overproduction of this protein and the accumulation of mature small subunits of mitochondrial ribosomes indicate that the cyt-U-28 mutant preferentially expresses two mitochondrial genes, one coding for ATPase subunit 9, the other for the S5 ribosomal protein.Key words: Neurospora, mitochondria, ribosomes, protein synthesis.

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than ;mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.

Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein.

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.

Light-dependent assembly of ribulose-1,5-bisphosphate carboxylase

Ribulose-1,5-bisphosphate carboxylase [RuP(2)Case; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is composed of eight small subunits (M(r), 14,000) and eight large subunits (M(r), 55,000). Newly synthesized large subunits are associated with two complexes having sedimentation coefficients of 7 and 29 S. Assembly of RuP(2)Case occurs in isolated intact chloroplasts in the light but not in the dark. When extracts of chloroplasts are treated with ATP or GTP, RuP(2)Case assembly is accelerated while the 29S large subunit complex is maintained. In the presence of Mg(2+), ATP brings about almost complete dissociation of the 29S complex, whereas GTP and a nonhydrolyzable analog of ATP are without effect. These results indicate the existence of a complex set of reactions involving nucleotides, Mg(2+), and several putative intermediates in RuP(2)Case assembly. It is postulated that these reactions at least partly account for the light dependence of RuP(2)Case assembly. In particular, ATP and GTP promote the assembly of large subunits into RuP(2)Case.

Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts.

Ribulose bisphosphate carboxylase consists of cytoplasmically synthesized "small" subunits and chloroplast-synthesized "large" subunits. Large subunits of ribulose bisphosphate carboxylase synthesized in vivo or in organello can be recovered from intact chloroplasts in the form of two different complexes with sedimentation coefficients of 7S and 29S. About one-third to one-half of the large subunits synthesized in isolated chloroplasts are found in the 7S complex, the remainder being found in the 29S complex. Upon prolonged illumination of the chloroplasts, newly synthesized large subunits accumulate in the 18S ribulose bisphosphate carboxylase molecule and disappear from both the 7S and the 29S large subunit complexes. The 29S complex undergoes an in vitro dissociation reaction and is not as stable as ribulose bisphosphate carboxylase. The data indicate that (a) the 7S large subunit complex is a chloroplast product, the (b) the 29S large subunit complex is labeled in vivo, that (c) each of these two complexes can account quantitatively for all the large subunits assembled into RuBPCase in organello, and that (d) excess large subunits are degraded in chloroplasts.

Protein synthesis in chloroplasts IX. Assembly of newly-synthesized large subunits into ribulose bishopshate carboxylase in isolated intact pea chloroplasts

Isolated pea ( Pisum sativum) chloroplasts incorporate [ 35S]methionine into the large subunit of the chloroplast enzyme ribulose bisphosphate carboxylase. When chloroplasts are incubated in a medium containing KCl as osmoticum, newly-synthesised large subunits are not incorporated into the holoenzyme but can be separated from pre-existing enzyme by gel electrophoresis under non-denaturing conditions. Furthermore, newly-synthesised large subunits are not precipitated by antibodies which precipitate pre-existing holoenzyme and large subunit prepared from holoenzyme. When chloroplasts are incubated in a medium containing sorbitol as osmoticum, some of the newly-synthesised large subunits comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels. Such comigrating large subunits are precipitated by antibodies raised against the holoenzyme. These results indicate assembly of large subunits into ribulose bisphosphate carboxylase in the sorbitol medium. Time course experiments indicate that there is a time-lag of several minutes between the onset of synthesis of large subunits and the onset of assembly. Newly-synthesised large subunits which do not comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels are associated with a protein of subunit molecular weight 60 000. This protein may be specifically combined with newly-synthesised large subunits, and the resulting aggregate be involved in the assembly of complete molecules of ribulose bisphosphate carboxylase.