A subunit interaction in chloroplast ATP synthase determined by genetic complementation between chloroplast and bacterial ATP synthase genes

F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.     

Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin

We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.     

A 37.5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes, a TCP-1-related gene, an open reading frame similar to the DAL80 gene, and a tRNA(Arg)

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromosome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding gamma-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Cc eta subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA(Arg3) (AGC) gene.     

Isolation and characterization of the mitochondrial ATP synthase from Chlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the alpha subunit

We have isolated the F0F1-ATP synthase complex from oligomycin-sensitive mitochondria of the green alga Chlamydomonas reinhardtii. A pure and active ATP synthase was obtained by means of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and PMSF [corrected]. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase alpha subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58-70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase beta subunit from C. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771-780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15-18 residues longer than in ATP synthase alpha subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.     

The chaperonin of the archaeon Sulfolobus solfataricus is an RNA-binding protein that participates in ribosomal RNA processing

The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double-ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so-called CCT-TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exists that the archaeal chaperonins are implicated in protein folding, much remains to be learned about their precise cellular function. In this work, we report that the chaperonin of the thermophilic archaeon Sulfolobus solfataricus is an RNA-binding protein that interacts specifically in vivo with the 16S rRNA and participates in the maturation of its 5' extremity in vitro. We further show that the chaperonin binds RNA as the native heterooligomeric complex and that RNA binding and processing are inhibited by ATP. These results agree with previous reports indicating a role for the bacterial/organellar chaperonins in RNA protection or processing and suggest that all known chaperonin families share specific and evolutionarily ancient functions in RNA metabolism.     

Group II chaperonin in a thermophilic methanogen, Methanococcus thermolithotrophicus. Chaperone activity and filament-forming ability

A gene encoding 544 amino acids for a subunit of group II chaperonin (thermosome) was cloned from a thermophilic methanogen, Methanococcus thermolithotrophicus. The deduced amino acid sequence showed 66.5, 56.1, and 20.1% similarities to those of Methanopyrus kandleri and Thermoplasma acidophilum and group I chaperonin of Escherichia coli, respectively. We call this chaperonin MTTS (M. thermolithotrophicus thermosome). The MTTS gene was expressed in E. coli. The purified recombinant MTTS seemed to be monomeric on gel filtration in the absence of Mg2+ and ATP. The monomer assembled to an oligomer (complex) in the presence of 50 mM MgCl2, 0.25 mM ATP, and 0.3 M (NH4)2SO4. It was eluted immediately before the elution volume of E. coli GroEL tetradecamer on gel filtration with a TSKgel G3000SWXL column. This reconstructed MTTS complex showed the cylindrical structure with two stacked rings in electron microscopy. The MTTS complex formed filamentous structures in the presence of Mg2+ and ATP at the protein concentration above 3.0 mg/ml. This filament formation was reversible. The MTTS filament was dissociated to the complex by dilution to the protein concentration of 0.2 mg/ml, even in the presence of Mg2+ and ATP. The MTTS complex exhibited weak ATPase activity with the hydrolysis rate of 74 mol of ATP hydrolysis/mol of MTTS complex/min at 70 degreesC. The MTTS complex promoted the refolding of chemically denatured thermophilic archaeal citrate synthase and glucose dehydrogenase at 50 degreesC in an ATP-dependent fashion. The analysis of nucleotide specificity of chaperone activity of MTTS suggested that it was coupled with hydrolysis of ATP, CTP, or UTP.     

Prevention of in vitro protein thermal aggregation by the Sulfolobus solfataricus chaperonin. Evidence for nonequivalent binding surfaces on the chaperonin molecule

We have studied the effects of the Sulfolobus solfataricus chaperonin on the aggregation and inactivation upon heating of four model enzymes: chicken egg white lysozyme (one 14.4-kDa chain), yeast alpha-glucosidase (one 68.5-kDa chain), chicken liver malic enzyme (four 65-kDa subunits), and yeast alcohol dehydrogenase (four 37.5-kDa subunits). When the proteins were heated in the presence of an equimolar amount of chaperonin, 1) the aggregation was prevented in all solutions; 2) the inactivation profiles of the single-chain enzymes were comparable with those detected in the absence of the chaperonin, and enzyme activities were regained in the solutions heated in the presence of the chaperonin upon ATP hydrolysis (78 and 55% activity regains for lysozyme and alpha-glucosidase, respectively); 3) the inactivation of the tetrameric enzymes was completely prevented, whereas the activities decreased in the absence of the chaperonin. We demonstrate by gel filtration chromatography that the chaperonin interacted with the structures occurring during thermal denaturation of the model proteins and that the interaction with the single-chain proteins (but not that with the tetrameric proteins) was reversed upon ATP hydrolysis. The chaperonin had nonequivalent surfaces for the binding of the model proteins upon heating: the thermal denaturation intermediates of the single-chain proteins share Surfaces I, while the thermal denaturation intermediates of the tetrameric proteins share Surfaces II. ATP binding to the chaperonin induced a conformation that lacked Surfaces I and carried Surfaces II. These data support the concept that chaperonins protect native proteins against thermal aggregation by two mechanistically distinct strategies (an ATP-dependent strategy and an ATP-independent strategy), and provide the first evidence that a chaperonin molecule bears functionally specialized surfaces for the binding of the protein substrates.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

Selected subunits of the cytosolic chaperonin associate with microtubules assembled in vitro

The molecular chaperone activities of the only known chaperonin in the eukaryotic cytosol (cytosolic chaperonin containing T-complex polypeptide 1 (CCT)) appear to be relatively specialized; the main folding substrates in vivo and in vitro are identified as tubulins and actins. CCT is unique among chaperonins in the complexity of its hetero-oligomeric structure, containing eight different, although related, gene products. In addition to their known ability to bind to and promote correct folding of newly synthesized and denatured tubulins, we show here that CCT subunits alpha, gamma, zeta, and theta also associated with in vitro assembled microtubules, i.e. behaved as microtubule-associated proteins. This nucleotide-dependent association between microtubules and CCT polypeptides (Kd approximately 0.1 microM CCT subunit) did not appear to involve whole oligomeric chaperonin particles, but rather free CCT subunits. Removal of the tubulin COOH termini by subtilisin digestion caused all eight CCT subunits to associate with the microtubule polymer, thus highlighting the non-chaperonin nature of the selective CCT subunit association with normal microtubules.     

A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2

A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta').     

Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I

In vivo, nucleosomes are formed rapidly on newly synthesized DNA after polymerase passage. Previously, a protein complex from human cells, termed chromatin assembly factor-I (CAF-I), was isolated that assembles nucleosomes preferentially onto SV40 DNA templates that undergo replication in vitro. Using a similar assay, we now report the purification of CAF-I from the budding yeast Saccharomyces cerevisiae. Amino acid sequence data from purified yeast CAF-I led to identification of the genes encoding each subunit in the yeast genome data base. The CAC1 and CAC2 (chromatin assembly complex) genes encode proteins similar to the p150 and p60 subunits of human CAF-I, respectively. The gene encoding the p50 subunit of yeast CAF-I (CAC3) is similar to the human p48 CAF-I subunit and was identified previously as MSI1, a member of a highly conserved subfamily of WD repeat proteins implicated in histone function in several organisms. Thus, CAF-I has been conserved functionally and structurally from yeast to human cells. Genes encoding the CAF-I subunits (collectively referred to as CAC genes) are not essential for cell viability. However, deletion of any CAC gene causes an increase in sensitivity to ultraviolet radiation, without significantly increasing sensitivity to gamma rays. This is consistent with previous biochemical data demonstrating the ability of CAF-I to assemble nucleosomes on templates undergoing nucleotide excision repair. Deletion of CAC genes also strongly reduces silencing of genes adjacent to telomeric DNA; the CAC1 gene is identical to RLF2 (Rap1p localization factor-2), a gene required for the normal distribution of the telomere-binding Rap1p protein within the nucleus. Together, these data suggest that CAF-I plays a role in generating chromatin structures in vivo.     

Chromophore-assisted laser inactivation of subunits of the T-cell receptor in living cells is spatially restricted

Chromophore-assisted laser inactivation (CALI) is a molecular photoablation technique that has been used to elucidate the in vivo roles of specific proteins in neural development. The interpretation of its effects on proteins in living cells relies on knowing how spatially restricted the CALI-induced damage is in vivo. To determine the spatial specificity of CALI in living cells, we have applied CALI to individual subunits of the T-cell receptor (TCR) complex on the surface of 2B4 hybridoma cells in culture and have examined the consequent structural and functional integrity of the TCR-alpha, TCR-beta and CD3-epsilon. The CALI of TCR-beta resulted in the disruption of the beta subunit and also resulted in a small effect on antibody binding alone to the neighboring TCR-alpha but caused no effect on another subunit, CD3-epsilon. Reciprocal experiments directing CALI to TCR-alpha and CD3-epsilon gave consistent results. No effects other than a simple loss of function were observed for any of these CALI experiments. These data demonstrate the extent of CALI-induced damage within a multisubunit complex in living cells and provide greater confidence for the future application of this technique to understanding in vivo function of proteins during complex cellular processes.     

The human glycine receptor beta subunit: primary structure, functional characterisation and chromosomal localisation of the human and murine genes

The inhibitory glycine receptor (GlyR) is a pentameric receptor comprised of alpha and beta subunits, of which the beta subunit has not been characterised in humans. A 2106 bp cDNA, isolated from a human hippocampal cDNA library, contained an open reading frame of 497 amino acids which encodes the beta subunit of the human GlyR. The mature human GlyR beta polypeptide displays 99% amino acid identity with the rat GlyR beta subunit and 48% identity with the human GlyR alpha 1 subunit. Neither [3H]strychnine binding nor glycine-gated currents were detected when the human GlyR beta subunit cDNA was expressed in the human embryonic kidney 293 cell line. However, co-expression of the beta subunit cDNA with the alpha 1 subunit cDNA resulted in expression of functional GlyRs which showed a 4-fold reduction in the EC50 values when compared to alpha 1 homomeric GlyRs. Glycine-gated currents of alpha 1/beta GlyRs were 17-fold less sensitive than homomeric alpha 1 GlyRs to the antagonists picrotoxin, picrotoxinin and picrotin, providing clear evidence that heteromeric alpha 1/beta GlyRs were expressed. The beta subunit appears to play a structural rather than ligand binding role in GlyR function. Fluorescence in situ hybridisation was used to localise the gene encoding the human GlyR beta subunit (GLRB) to chromosome 4q32, a position syntenic with mouse chromosome 3. In situ hybridisation using the human GlyR beta subunit cDNA showed that the murine GlyR beta subunit gene (Glrb) maps to the spastic (spa) locus on mouse chromosome 3 at bands E3-F1. This is consistent with the recent finding that a mutation in the murine GlyR beta subunit causes the spa phenotype. It also raises the possibility that mutations in the human beta subunit gene may cause inherited disorders of the startle response.     

Detection of a homozygous four base pair deletion in the protein X gene in a case of pyruvate dehydrogenase complex deficiency

While the presence of a lipoyl-containing protein (protein X) separate from lipoyl transacetylase in the pyruvate dehydrogenase complex (PDC) has been known for some time, until recently only the cDNA for the yeast enzyme has been cloned. We have cloned, sequenced and characterized the cDNA encoding the human protein X and localized the protein X gene to chromosome 11p13. We also report here a new case of protein X deficiency identified immunologically, with decreased activity of PDC and without mutations in the E1alpha subunit or E1beta subunit. We report that the cDNA and gene of this patient for protein X has a homozygous 4 bp deletion, specifically in the putative mitochondrial targeting signal sequence which results in a premature stop codon. This is the first documented case of a molecular defect in pyruvate dehydrogenase protein X.     

The internal workings of a DNA polymerase clamp-loading machine

Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader. We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The gamma complex uses ATP to open the beta clamp and assemble it onto DNA. Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp. The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it. The delta' subunit acts as a modulator of the interaction between delta and beta. On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface. The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex.     

Purification of the pyruvate dehydrogenase multienzyme complex of Zymomonas mobilis and identification and sequence analysis of the corresponding genes

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1alpha (38 kDa) and E1beta (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome of Z. mobilis. The genes pdhAalpha (1,065 bp) and pdhAbeta (1,389 bp), encoding the E1alpha and E1beta subunits of the E1 component, were located downstream of the gene encoding enolase. The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhAalpha, pdhAbeta, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of the pdhB gene contains a single lipoyl domain. The E1beta subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.     

Qigong Yangsheng as a complementary therapy in the management of asthma: a single-case appraisal

In Caucasians, a genetic polymorphism is known for some alcohol dehydrogenase (ADH) systems (beta and gamma). Knowledge of population frequencies of the beta and gamma hepatic isozyme variants in France is a prerequisite to understanding any role this genetic variation might play in determining the risk of alcohol-related diseases. For the present study, the ADH phenotypes of 115 French Caucasian control subjects, consisting of 64 men and 51 women, were studied. The beta2 subunit was found to have a very low frequency. The relative frequencies of ADH gamma subunits observed in French Caucasians were found to be in good agreement with those already observed in other Caucasian populations. When gender and age were taken into account, a particular group corresponding to young men (age <50 years) was characterized by a manifest discordance with the comparable female population.     

Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.     

Characterization of DNA sequences encoding a novel isoform of the 55 kDa B regulatory subunit of the type 2A protein serine/threonine phosphatase of Arabidopsis thaliana

We have identified a novel gene (AtB beta) encoding a previously uncharacterized isoform of the B regulatory subunit of the type 2A serine/threonine protein phosphatase (PP2A) of Arabidopsis, and show that mRNA derived from the AtB beta gene accumulates in all Arabidopsis organs. In addition, we examined the expression of the three genes encoding the A regulatory subunit of Arabidopsis PP2A and show these genes are expressed in all organs as well. Taken together, our results suggest a myriad of PP2A subunit combinations, possibly with distinct substrate specificities, may occur within each Arabidopsis cell.