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PubMed2000

Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis.

Cell division in many eukaryotes, including the fission yeast Schizosaccharomyces pombe, utilizes a contractile actomyosin ring. In S. pombe, the actomyosin ring is assembled at the medial cortex upon entry into mitosis and constricts at the end of anaphase to guide the centripetal deposition of the septum. Despite identification of several structural components essential for actomyosin ring assembly, the interdependencies between these gene-products in the process of ring assembly are unknown. This study investigates the role of Rng3p, a member of the UCS-domain containing protein family (Unc-45p, Cro1p, She4p), in actomyosin ring assembly. Null mutants in rng3 resemble deletion mutants in the type II myosin heavy chain (myo2) and rng3(ts) mutants show strong negative interactions with the myo2-E1 mutant, suggesting that Rng3p is involved in modulating aspects of type II myosin function. Interestingly, a green fluorescent protein (GFP) tagged Rng3p fusion is detected at the division site in the myo2-E1 mutant, but not in other myo2-alleles, wild-type cells or in 18 other cytokinesis mutants. Assembly and maintenance of Rng3p at the division site in the myo2-E1 mutant requires F-actin. Rng3p is also required for the proper assembly of Myo2p and F-actin into a functional actomyosin ring but is not necessary for their accumulation at the division site. We conclude that Rng3p is a novel component of the F-actin cytoskeleton essential for a late step in actomyosin ring assembly and that it might monitor some aspect of type II myosin assembly during actomyosin ring construction.

Actomyosin↗
PubMed2000

A novel RGD-independent fibronectin assembly pathway initiated by alpha4beta1 integrin binding to the alternatively spliced V region.

Fibronectin (FN) matrix assembly is a multi-step process that involves binding to integrin receptors, FN-FN interactions and connections to the actin cytoskeleton. Ultimately, FN is converted into stable matrix fibrils that are detergent-insoluble. RGD-binding integrins such as alpha5beta1 play a major role in the assembly of fibrillar FN. Here we show that alpha4beta1 binding to the alternatively spliced V (IIICS) region of FN initiates an alternative assembly pathway. Activation of alpha4beta1 with exogenous agents such as Mn(2+) or a beta1-stimulatory antibody TS2/16 was sufficient to induce initiation of FN fibrillogenesis by Ramos B lymphoma cells and by CHO(B2)alpha4 cells. Using recombinant FNs lacking specific sequences, we show that assembly is independent of the RGD sequence but requires the V25/CS-1 segment. Previously, we have characterized an activated recombinant FN (FN III(1-7)) that rapidly forms detergent-insoluble multimers upon binding to alpha5beta1 integrin. Alpha4beta1 also formed FNdeltaIII(1-7) multimers without the aid of exogenous stimulants, suggesting that an activated form of FN can override the need for activation of the integrin. In contrast to assembly by alpha5beta1, actin filaments remained largely cortical and no change in cell growth rate was observed with alpha4beta1-mediated assembly. These results show that binding sites on FN other than the RGD sequence/synergy site and distant from the cell binding domain can promote FN assembly. Thus, there appear to be multiple, integrin-specific mechanisms for assembly of FN matrix.

Alternative Splicing↗
PubMed1993

Assembly of envelope structure with vesicles associated with Ku-homologous protein in Xenopus egg extract in the absence of chromatin.

To study the process of nuclear envelope assembly at the end of mitosis, we developed a chromatin-free in vitro system for assembly of envelope structures in Xenopus interphase egg extract, and examined the participation of Ku-homologous protein in the assembly. The envelope structure assembled spontaneously in the absence of chromatin or DNA between glass plates under a condition that minimized generation of flow of the extract. Morphological study using an electron microscopy has revealed that the membrane surrounding the envelope structure is a double membrane that contains gaps resembling nuclear pore complex. Their assembly was dependent on ATP and was inhibited by the addition of GTP-gamma-S or N-ethylmaleimide. Depletion of a pre-nuclear vesicle by preincubating the interphase egg extracts with large excess of sperm head chromatins impaired the assembly. The membrane vesicle, which was associated with Ku-homologous protein of Xenopus, participated in the assembly as proven by reaction with monoclonal antibody made specific for Ku p70 protein. However, the assembly process of the envelope structure was inhibited only slightly by the antibody, suggesting that the Ku-homologous protein does not participate in the fusion process of vesicles to form the envelope structure.

Adenosine Triphosphate↗
PubMed2024

Taking advantage of reference-guided assembly in a slowly-evolving lineage: Application to Testudo graeca.

BACKGROUND: Obtaining de novo chromosome-level genome assemblies greatly enhances conservation and evolutionary biology studies. For many research teams, long-read sequencing technologies (that produce highly contiguous assemblies) remain unaffordable or unpractical. For the groups that display high synteny conservation, these limitations can be overcome by a reference-guided assembly using a close relative genome. Among chelonians, tortoises (Testudinidae) are considered one of the most endangered taxa, which calls for more genomic resources. Here we make the most of high synteny conservation in chelonians to produce the first chromosome-level genome assembly of the genus Testudo with one of the most iconic tortoise species in the Mediterranean basin: Testudo graeca. RESULTS: We used high-quality, paired-end Illumina sequences to build a reference-guided assembly with the chromosome-level reference of Gopherus evgoodei. We reconstructed a 2.29 Gb haploid genome with a scaffold N50 of 107.598 Mb and 5.37% gaps. We sequenced 25,998 protein-coding genes, and identified 41.2% of the assembly as repeats. Demographic history reconstruction based on the genome revealed two events (population decline and recovery) that were consistent with previously suggested phylogeographic patterns for the species. This outlines the value of such reference-guided assemblies for phylogeographic studies. CONCLUSIONS: Our results highlight the value of using close relatives to produce de novo draft assemblies in species where such resources are unavailable. Our annotated genome of T. graeca paves the way to delve deeper into the species' evolutionary history and provides a valuable resource to enhance direct conservation efforts on their threatened populations.

Animals↗
PubMed2004

Assembly of plant Shaker-like K(out) channels requires two distinct sites of the channel alpha-subunit.

SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K(+) channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K(out) channels. Deletion mutants and chimeric proteins generated from SKOR and the K(in) channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K(T) domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K(out) alpha-subunits did not assemble with K(in) alpha-subunits because of the absence of interaction between their assembly sites.

Animals↗
PubMed2005

The SC3 hydrophobin self-assembles into a membrane with distinct mass transfer properties.

Hydrophobins are a class of small proteins that fulfill a wide spectrum of functions in fungal growth and development. They do so by self-assembling into an amphipathic membrane at hydrophilic-hydrophobic interfaces. The SC3 hydrophobin of Schizophyllum commune is the best-studied hydrophobin. It assembles at the air-water interface into a membrane consisting of functional amyloid fibrils that are called rodlets. Here we examine the dynamics of SC3 assembly at an oil-water and air-water interface and the permeability characteristics of the assembled layer. Hydrophobin assembled at an oil-water interface is a dynamic system capable of emulsifying oil. It accepts soluble-state SC3 oligomers from water in a unidirectional process and sloughs off SC3 vesicles back into the water phase enclosing a portion of the oil phase in their hydrophobic interior. The assembled layer is impermeable to solutes >200 Da from either the water phase or the oil phase; however, due to the emulsification process, oil and the hydrophobic marker molecules in the oil phase can be transferred into the water phase, thus giving the impression that the assembled layer is permeable to the marker molecules. By contrast, the layer assembled at an air-water interface is permeable to water vapor from either the hydrophobic or hydrophilic side.

Air↗
PubMed2005

Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae.

The spindle assembly checkpoint regulates the metaphase-to-anaphase transition from yeast to humans. We examined the genetic interactions with four spindle assembly checkpoint genes to identify nonessential genes involved in chromosome segregation, to identify the individual roles of the spindle assembly checkpoint genes within the checkpoint, and to reveal potential complexity that may exist. We used synthetic genetic array (SGA) analysis using spindle assembly checkpoint mutants mad1, mad2, mad3, and bub3. We found 228 synthetic interactions with the four spindle assembly checkpoint mutants with substantial overlap in the spectrum of interactions between mad1, mad2, and bub3. In contrast, there were many synthetic interactions that were common to mad1, mad2, and bub3 that were not shared by mad3. We found shared interactions between pairs of spindle assembly checkpoint mutants, suggesting additional complexity within the checkpoint and unique interactions for all of the spindle assembly checkpoint genes. We show that most genes in the interaction network, including ones with unique interactions, affect chromosome transmission or microtubule function, suggesting that the complexity of interactions reflects diverse roles for the checkpoint genes within the checkpoint. Our analysis expands our understanding of the spindle assembly checkpoint and identifies new candidate genes with possible roles in chromosome transmission and mitotic spindle function.

Cell Cycle Proteins↗
PubMed2026

Cooperation of transposable elements to endow global networks of initiators of hybrid assembly pathways of endogenous multiprotein complexes.

Mechanisms governing initiation steps of the assembly of endogenous multi-protein complexes (EMC) remain incompletely understood. Here, multiple lines of observations are reported describing the function-aligned initiation sequence of hybrid assembly pathways (HAP) of EMC. The first step of HAP-guided chain reactions of protein-protein interactions (PPI) of EMC assemblies constitutes the creation of cell type-specific pools of hetero and homo dimers. The molecular anatomy of HAP was elucidated by defining qualitative and quantitative characteristics of protein binding to a compendium of 200,393 distinct genomic regulatory elements (GRE), including 49,667 sequences representing control sets of genomic loci as well as 150,726 GRE of different evolutionary origins. The consensus sequence of HAP actions consists of: a) Initiation on genomic DNA of the formation of metastable hetero- and homodimers of EMCs' protein constituents; b) Release of dimers from DNA templates for delivery to the EMC assembly compartments; c) Assembly of defined EMC by sequential on demand addition of proteins to preformed dimers serving as attractors of EMC-specific ensembles of monomers. Chromosome-naïve DNA scaffolds facilitating creation of intracellular dimer pools engage networks of ~700 transcription factors (TFs), 534 of which manifest region-specific patterns of significantly enriched expression in 1358 brain regions. HAP initiators appear to operate within nucleosome-depleted islands of transposable elements (TE) - derived sequences within heterochromatin. PPI assembly lines of EMCs operate in 2 concurrent modes: TF-TF PPI cascade and PPI HUB protein cascade. Regardless of the number of DNA-bound initiator TFs (ranging from one to 716 TFs), both modes of operations reached the equilibrium at the PPI constituents saturation levels of ~245 proteins for TF-TF PPI modes and of ~351 proteins for PPI HUB protein modes. Distinct panels of DNA-bound initiator TFs and proteins of PPI cascade ensembles are enriched in either defined sets of neuroanatomical structures (TF-TF mode) or among structural-functional constituents of synapses (HUB proteins mode). Thus, these bifurcated cascades appear biologically congruent: TF-TF constituents map to transcriptional signatures of hundreds of brain regions, whereas HUB constituents map to synaptogenesis and synaptic structures, suggesting the unified logic of genomic functions coordinating region identity and connectivity. Evidence-supported examples of default operations of PPI-guided assemblies of hetero- and homodimers of Yamanaka factors, neurogenesis constituents, and protein components of postsynaptic density of excitatory and inhibitory synaptogenesis are reported with detailed analytical focus on human Claustrum. The foundational set of observations reported in this contribution should facilitate experimental and theoretical explorations of TE-seeded genomic codes for initiators of PPI chain reactions of protein dimerization creating pools of attractors to guide and accelerate the EMC assemblies.

Humans↗
PubMed2026

ImpuT2T: Pangenome-Based Patching for Human Genome Assemblies.

With improvements in sequencing and assembly have come many high-quality telomere-to-telomere assemblies and reference pangenomes. However, the long-read sequencing recipes needed for high quality assemblies are expensive, and out of reach for many research groups. Here we propose ImpuT2T, a method that takes an assembly produced via inexpensive HiFi sequencing reads, and uses a panel of T2T (or near-T2T) assemblies to scaffold and fill ("patch") the gaps between the HiFi contigs. Benchmarking against reference assemblies demonstrates that ImpuT2T is highly effective at patching human HiFi assemblies, consistently outperforming existing patching approaches. Moreover, we show that including more haplotypes in the pangenome improves the quality of the patched assemblies, with the greatest gains achieved using the full HPRC Release 2 pangenome.

Journal Article↗
PubMedDate not supplied

[In vitro self assembly of supermolecular structures after spontaneous disassembly of carotovoricins].

The self-assembly of supramolecular structures (empty sheaths and polysheaths of the macromolecular Erwinia carotovora bacteriocins) was studied by electron microscopy in the course of 1- to 2-year incubation of phage particles at 4 degrees C. This study showed that the empty sheaths and polysheaths of the bacteriocins of eight E. carotovora strains spontaneously assemble at the self-assembly centers (or crystallization centers), which have a diameter of 26-65 nm and contain a dense proteinaceous material. The self-assembly center consists of two components, a primer and the structural protein of contracted sheaths. Empty sheaths assembled in the crystallization centers are polar structures synthesized through the stepwise head-to-tail polymerization of monomeric units. The supramolecular structures of two E. carotovora 62A bacteriocins are assembled in a different way. At the early stages of their self-assembly, a reticular structure is formed, which then transforms into very long polysheaths composed of monomers. Along with polysheaths, rounded or lamplike structures 33-117 nm in size composed of the subunits of contracted sheath are produced. Carotovoricins may serve as suitable objects for the study of the self-assembly of elementary biological structures.

Bacteriocins↗
PubMed2003

Amelogenin self-assembly and the role of the proline located within the carboxyl-teleopeptide.

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.

Amelogenesis↗
PubMed1992

Subunit interactions involved in the assembly of pyridine nucleotide transhydrogenase in the membranes of Escherichia coli.

The pyridine nucleotide transhydrogenase (PNT) of Escherichia coli consists of two different subunits (alpha and beta) and assembles as a tetramer (alpha 2 beta 2) in the inner membrane. The pnt genes from E. coli have been cloned on a multicopy plasmid resulting in high level expression of the enzyme activity. We have studied the influence of the different segments of the polypeptide chains of the alpha and beta subunits on the assembly and function of the enzyme by constructing a series of deletion mutants for both of the subunits. Our results show that the assembly of the beta subunit is contingent upon the insertion of the alpha subunit into the membrane, while the alpha subunit can assemble independently of the beta subunit. All deletions constructed for the cytosolic portion of the alpha subunit gave no incorporation of the alpha subunit and, as a consequence, of the beta subunit, also. Of the four membrane-spanning regions of the alpha subunit, the last two were indispensable, while the deletion of the first two still allowed the association of alpha as well as of the beta subunit with the membrane. However, the enzyme was not functional. The two subunits were also loosely associated as mild detergent treatment released them from the membrane in contrast with the wild-type enzyme. Deletions within the beta subunit had little effect on the assembly of the alpha subunit, although less was incorporated. All deletions involving the cytosolic portion of the beta subunit resulted in loss of incorporation into the membrane. Of the eight membrane-spanning regions of the beta subunit, the deletion of regions 2-3, 2-4, 2-6, and 2-7 yielded significant association of both the subunits with the membrane. However, none of these mutants assembled a functional enzyme, and again the two subunits were loosely associated with the membrane. Based on the stringent requirement of the cytosolic portions of alpha and beta subunits for assembly, a model is proposed that suggests interactions between these two regions must occur prior to assembly.

Cell Membrane↗
PubMed1991

Effects of retinal detachment on rod disc membrane assembly in cultured frog retinas.

The authors compared rod outer segment (ROS) disc membrane assembly rates in detached and attached frog retinas to determine if there was a rapid impairment of membrane assembly in response to retinal detachment. Membrane assembly was quantified in vitro by incubating retinas in medium containing Lucifer yellow, which is entrapped by nascent discs. Video microscopy was used to detect incorporation of the dye. During the first 10 hr after separation of the retina from the retinal pigment epithelium (RPE), ROS-disc membrane assembly in isolated Xenopus laevis neural retinas continued at a near normal rate, 0.81 microns/10 hr, a 13% reduction (P less than .01), compared with the 0.93 microns/10 hr observed in attached control retinas. The morphology of the OS appeared normal in most rod photoreceptors by transmission electron microscopy, although vesiculation of the most basal OS membranes was seen in a small population (25%) of rods. Approximately 90% of rod photoreceptors continued to assemble OS membranes for more than 10 hr after detachment, but by the end of 2 days, only 55% were still making new discs. The percentage of rods with normal basal OS membranes also decreased (to approximately 50%). Therefore, only 25% were assembling morphologically normal discs 2 days after detachment. In attached control regions, rod photoreceptors showed a comparatively minor response to culture conditions; assembly of morphologically normal discs continued for 2 days in about 85% and ceased in only 10%. These results indicate that the effects on disc membrane assembly of disrupting photoreceptor-RPE interaction in vitro initially are slight but become progressively severe with time.

Animals↗
PubMed1989

Lamin B methylation and assembly into the nuclear envelope.

Lamin B is reversibly methyl-esterified and phosphorylated during the mammalian cell cycle. In order to study the role of methylation in lamin B function, we isolated mitotic cells in the presence of the microtubule inhibitor, nocodazole. Following removal of nocodazole, methylation of mitotic lamin B was found to precede its assembly into the nuclear envelope as cells exited mitosis. Very little additional methylation took place on assembled lamins. We were able to slow the rate of lamin B methylation with methylthioadenosine (MTA). A delay in lamin B methylation was accompanied by a corresponding delay in assembly of lamin B into the envelope. The delay was approximately 20-30 min beyond the typical 60-70 min usually required. Assembly of lamins A and C, which are not methylated, was also delayed by MTA, although to a lesser degree, suggesting that an interaction between the lamins is necessary for formation of the nuclear envelope. Chromatin decondensation was also slowed in the presence of MTA. Other inhibitors of methylation which had no inhibitory effect on the methyl esterification of lamin B were tested and found to have no effect on envelope assembly or chromatin decondensation. These results were obtained with Chinese hamster ovary cells as well as with the stem cell line, PC 13. Dephosphorylation of lamin B normally follows a time course similar to that of nuclear envelope assembly. In the presence of MTA, however, lamin B assembly was slowed with little effect on dephosphorylation. This resulted in a large population of dephosphorylated, but unassembled, lamin B protein, demonstrating that dephosphorylation is not sufficient for envelope assembly. The lack of effect on the time course of dephosphorylation also suggests that MTA is not acting upstream of the methylation event.

Adenosine↗
PubMed1981

Subunit of assembly of Escherichia coli RNA polymerase.

The isolated subunits of Escherichia coli DNA-dependent RNA polymerase are reassembled in a stepwise manner in the following sequence: 2 alpha leads to alpha 2 leads to alpha 2 beta leads to alpha 2 beta beta' (premature core enzyme) leads to E (active core enzyme). When the in vitro reconstitution is performed at low temperature, the subunit assembly is prevented until the assembled but inactive premature core enzyme is formed, which is similar to native core enzyme in many parameters of gross conformation but differs from it in several minor and local conformations. The temperature-dependent activation of premature core enzyme at a salt concentration as low as that in vivo takes place only in the continuous presence of either the sigma subunit or DNA. The sigma subunit is therefore proposed to be a regulatory protein which influences the conformation of core subunit assembly in multiple ways from the initial enzyme maturation to the final initiation of transcription. Evidence has accumulated which indicates that the subunit assembly in vivo proceeds via the same pathway as that identified in vitro, including the identification of all species of the assembly intermediates in cell extracts, the identification of all possible types of assembly-defective mutants among temperature-sensitive alpha-, beta-, and beta'-subunit mutants, the kinetics of the appearance of pulse-labeled subunits in the enzyme structure as expected from the assembly sequence and the integration of labeled subassemblies into the enzyme structure upon chasing. The functional complexity of RNA polymerase coupled with transcriptional control appears to depend on its structural flexibility which fluctuates through the assembly with various transcription factors. This type of transcriptional control is being thoroughly considered by a final conclusion awaits further examinations.

DNA-Directed RNA Polymerases↗
PubMed1982

Effect of ATP on the kinetics of microtubule assembly.

We investigated the role of ATP in the assembly of microtubules. Tubulin, prepared by chromatography on DEAE-cellulose, was nearly devoid of nucleoside diphosphokinase activity. ATP induced assembly in such preparations for a single assembly/disassembly cycle; then further assembly could not be induced by ATP unless the system was supplemented with additional GTP. This suggests that the E-site must contain GTP for polymerization and ATP interacts at a different site on tubulin. Although tubulin can be assembled into microtubules in 1.0 mM GTP, the inclusion of 0.2 mM ATP along with the GTP increases the rate and extent of assembly. The enhancement increased with increasing ATP concentrations. The inclusion of 0.2 mM ATP reduced the critical concentration for tubulin assembly from 1.5 to 0.9 mg/ml. Analysis of assembly rate versus protein concentration suggested that ATP also affects nucleation. Aggregates of tubulin rings formed by warming tubulin in the presence of 1.0 mM ATP and 5.0 mM Mg2+ were capable of initiating assembly in a solution of tubulin which was not able to polymerize. Furthermore, the extent of microtubule formation was dependent on the concentration of aggregated rings added to the solution. We propose that ATP interacts with tubulin at a binding site that is distinct from the N- and E-sites that bind GTP. A function of ATP binding is to stimulate the formation of tubulin rings as nucleation centers for polymerization.

Adenosine Triphosphate↗
PubMed1993

Assembly of the extracellular domain of the Na,K-ATPase beta subunit with the alpha subunit. Analysis of beta subunit chimeras and carboxyl-terminal deletions.

The role of the extracellular domain of the Na,K-ATPase beta subunit in assembly with the alpha subunit was investigated. A chimeric protein consisting of the extracellular domain of the beta subunit fused with the transmembrane and cytoplasmic domains of dipeptidyl peptidase IV assembles with the alpha subunit. An inverse chimera consisting of the cytoplasmic and transmembrane domains of the beta subunit fused with the extracellular domain of dipeptidyl peptidase IV does not assemble with the alpha subunit. The assembly data from these chimeras demonstrate that the extracellular domain of the beta subunit is both necessary and sufficient for assembly with the alpha subunit. Deletions of up to 146 extracellular amino acids from the carboxyl terminus of the beta subunit appear to result in misfolding of the subunit, but do allow reduced assembly with the alpha subunit. Together, the assembly data from chimeras and carboxyl-terminal deletions have identified a 96-residue extracellular domain which contains sequences involved in subunit assembly. While the chimeric subunits properly localize to the plasma membrane, deletion of as few as 4 amino acids from the carboxyl terminus impairs the ability of the beta subunit to be transported to the plasma membrane.

Amino Acid Sequence↗
PubMed1994

The effect of groES on the groEL-dependent assembly of dodecameric glutamine synthetase in the presence of ATP and ADP.

The yields of active dodecameric glutamine synthetase (GS) are significantly increased when in vitro folding is initiated in the presence of the Escherichia coli groE chaperonins and ATP (37 degrees C). To observe the effects of chaperonins and ATP on GS assembly, the GS assembly intermediates were separated by nondenaturing gel electrophoresis, visualized by Western analysis, and studied as a function of time. The form of GS that was initially released from groEL is monomeric. After the monomers formed dimers, active GS oligomers were assembled by the association of assembly competent dimers with higher order even-numbered oligomers until the dodecamer was formed. When ATP was added to the groEL.GS complex (no groES), a groEL.GS complex remained visible for up to 30 min after the renaturation was initiated. This slow disappearance of the groEL.GS complex is consistent with observed lags in both the GS activity regain profile and the assembly-dependent increase in GS tryptophan fluorescence. When groES was present, the addition of ATP resulted in the disappearance of observable complex at early sample times (< 2 min). Concomitantly, the rates of the regain of GS activity and the GS-dependent increase in tryptophan fluorescence intensity showed substantial accelerations. These results indicate that groES facilitates GS assembly from groEL by inducing the rapid release of GS from groEL, which in turn increases the concentration of assembly competent GS monomers. In addition, groES can initiate renaturation of GS from the groEL.GS arrested complex in the presence of ADP. When chaperonin-dependent GS renaturation was initiated with ATP or ADP (> or = 2 mM), the rates were identical. Since ATP hydrolysis is not absolutely required, the combined binding energies of groES and ATP (or ADP) appear to be sufficient to weaken the binding affinity of groEL for GS subunits and facilitate the release and refolding of assembly competent GS monomers from groEL.

Adenosine Diphosphate↗