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PubMed2001

TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.

In Escherichia coli, a subset of periplasmic proteins is exported via the twin-arginine translocation (Tat) pathway. In the present study, we have purified the Tat complex from E. coli, and we show that it contains only TatA, TatB, and TatC. Within the purified complex, TatB and TatC are present in a strict 1:1 ratio, suggesting a functional association. This has been confirmed by expression of a translational fusion between TatB and TatC. This Tat(BC) chimera supports efficient Tat-dependent export, indicating that TatB and TatC act as a unit in both structural and functional terms. The purified Tat complex contains varying levels of TatA, suggesting a gradual loss during isolation and a looser association. The molecular mass of the complex is approximately 600 kDa, demonstrating the presence of multiple copies of TatA, B, and C. Co-immunoprecipitation experiments show that TatC is required for the interaction of TatA with TatB, suggesting that TatA may interact with the complex via binding to TatC.

Base Sequence↗
PubMed1990

Occurrence in the archaebacterium Sulfolobus solfataricus of a ribosomal protein complex corresponding to Escherichia coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)2.P0.

Two-dimensional electrophoresis of total protein from 50 S ribosomal subunits of the archaebacterium Sulfolobus solfataricus demonstrated a complex between two proteins that was stable in 6 M urea, but dissociable in detergent or below pH 5.5. The proteins, numbered L1 and L10 according to their electrophoretic mobilities, corresponded to Escherichia coli ribosomal proteins L10 and L7/L12, respectively. The members of the complex were therefore designated Sso L10e and Sso L12e. Sso L12e had other properties in common with E. coli L7/L12: low molecular weight, relative acidity, selective release from the ribosome by high salt/ethanol, and dimeric structure. The Sso L12e.Sso L10e complex was isolated by gel filtration of total 50 S proteins in 4 M urea. The stoichiometry of the components was approximately four copies of Sso L12e to one copy of Sso L10e. The occurrence in an archaebacterium of a complex of acidic ribosomal proteins similar to E. coli (L7/L12)4.L10 and eukaryotic (P1)2/(P2)/.P0 strongly supports the concept that this element of quaternary structure is a major conserved feature of the ribosome and reaffirms its importance in the translocation step of protein synthesis.

Archaea↗
PubMed2005

Targeting the NAD7 subunit to mitochondria restores a functional complex I and a wild type phenotype in the Nicotiana sylvestris CMS II mutant lacking nad7.

The mitochondrial DNA of the Nicotiana sylvestris CMSII mutant carries a 72-kb deletion comprising the single copy nad7 gene that encodes the NAD7 subunit of the respiratory complex I (NADH-ubiquinone oxidoreductase). CMSII plants lack rotenone-sensitive complex I activity and are impaired in physiological and phenotypical traits. To check whether these changes directly result from the deletion of nad7, we constructed CMS transgenic plants (termed as CMSnad7) carrying an edited nad7 cDNA fused to the CAMV 35S promoter and to a mitochondrial targeting sequence. The nad7 sequence was transcribed and translated and the NAD7 protein directed to mitochondria in CMSnad7 transgenic plants, which recovered both wild type morphology and growth features. Blue-native/SDS gel electrophoresis and enzymatic assays showed that, whereas fully assembled complex I was absent from CMSII mitochondria, a functional complex was present in CMSnad7 mitochondria. Furthermore, a supercomplex involving complex I and complex III was present in CMSnad7 as in the wild type. Taken together, these data demonstrate that lack of complex I in CMSII was indeed the direct consequence of the absence of nad7. Hence, NAD7 is a key element for complex assembly in plants. These results also show that allotopic expression from the nucleus can fully complement the lack of a mitochondrial-encoded complex I gene.

Amino Acid Sequence↗
PubMed2001

GCP5 and GCP6: two new members of the human gamma-tubulin complex.

The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.

Amino Acid Sequence↗
PubMed1996

FliN is a major structural protein of the C-ring in the Salmonella typhimurium flagellar basal body.

The Salmonella typhimurium FliN protein has been proposed to form a mutually interacting complex with FliG and FliM, the switch complex, that is required for flagellar morphogenesis and function. We have used affinity chromatography for purification of extended flagellar basal bodies sufficient for quantitative analysis of their protein composition. The belled, extended structure is predominantly comprised of the switch complex proteins; with FliN present in the most copies (111 +/- 13). This explains why single, missense fliN, fliG or fliM mutations, found in many non-motile strains, can alter the belled morphology. Cell lysates from these strains contained the wild-type complement of FliG, FliM and FliN; but the basal bodies lacked the outer, cytoplasmic(C)-ring of the bell and were separated by sedimentation from FliM and FliN. The amount of FliG present in basal bodies from wild-type and one such mutant, FliN100LP, was comparable. These data show that: (1) the mutations define a FliG and FliMFliN multiple contact interface important for motility. (2) FliG is responsible for the increased size of the membrane-embedded MS-ring complex of belled relative to acid-treated basal bodies. (3) FliN, together with FliM, account for most of the C-ring. As a major component of the C-ring, FliN is distinct from the other proteins implicated in axial flagellar protein export. Inner, cytoplasmic rod basal substructure, seen by negative-stain and quick-freeze replica electron microscopy, may gate such export. Lack of connectivity between the cytoplasmic rod and ring substructures places contacts between FliG and FliMFliN at the periphery of the basal body, proximal to the flagellar intramembrane ring particles. This topology is consistent with models where torque results from interaction of circumferential arrays of the switch complex proteins with the ring particles.

Bacterial Proteins↗
PubMed1987

The organization and sequence of the genes for ATP synthase subunits in the cyanobacterium Synechococcus 6301. Support for an endosymbiotic origin of chloroplasts.

The nucleotide sequence has been determined of two regions of DNA cloned from the cyanobacterium Synechococcus 6301. The larger, 8890 base-pairs in length, contains a cluster of seven genes for subunits of ATP synthase. The order of the genes is a:c:b':b:delta:alpha:gamma, b' being a duplicated and diverged form of b. As in the Escherichia coli unc operon, the a gene is preceded by a gene for a small hydrophobic and basic protein. The hydrophobic profile of the potential gene product suggests that its secondary structure is similar to the uncI protein. The smaller DNA fragment, 4737 base-pairs in length, is separated from the larger by at least 15 X 10(3) base-pairs of DNA. It contains a cluster of two genes encoding ATP synthase subunits beta and epsilon. Both clusters of ATP synthase genes are preceded by sequences resembling the -10 (Pribnow) box of E. coli promoters and are followed by sequences able to form stable stem-loop structures that might serve to terminate transcription. These features and the small intergenic non-coding sequences suggest that the clusters are operons, for which the names atp1 and atp2 are proposed. The order of genes within the two clusters is very similar to the gene order in the E. coli unc operon. However, it is most closely related to the arrangement of genes for ATP synthetase subunits a:c:b:alpha and beta:epsilon in two clusters in pea chloroplast DNA. This close relationship between chloroplasts and the cyanobacterium is also evident from comparisons of the sequences of ATP synthase subunits; the Synechococcus proteins are much more closely related to chloroplast homologues than to those in other bacteria or in mitochondria. It is further supported by the cyanobacterial b and b' proteins which, in common with their chloroplast counterpart, subunit I, have extra amino-terminal extensions relative to the E. coli b protein. This extension is known to be removed by post-translational processing in the chloroplast, but its function is obscure. It also seems likely that the cyanobacterial and chloroplast ATP synthases have important similarities in subunit composition. For example, the presence of two related genes, b and b', in the cyanobacterium suggests that its ATP synthase is a complex of nine polypeptides, and that it may have single copies of related b and b' proteins rather than two copies of identical b subunits as found in the E. coli enzyme.4+off

ATP Synthetase Complexes↗
PubMed1996

Protein inheritance: lambda plasmid replication perpetuated by the heritable replication complex.

BACKGROUND: Replication of a plasmid derived from the Escherichia coli phage lambda initiates by binding of the lambda O protein initiator to the origin of lambda DNA replication, ori lambda. The lambda P protein participates in subsequent steps of assembly of the lambda replication complex. A function of lambda P required for replication complex assembly is inactivated at 43 degrees C by the ts1 mutation. RESULTS: We found that the lambda replication complex assembled at 30 degrees C survives the temperature upshift in lambda crotsPts1 plasmid-harbouring bacteria. We present several lines of evidence that in this system (in which the replication complex assembly does not occur), the replication complex assembled prior to the temperature upshift is inherited by one of two daughter plasmid copies at each replication round for more than 30 cell generations. The 'old' replication complex-driven replication is chloramphenicol-resistant and rifampicin-sensitive. This replication is dependent on lambda O and host dnaK, dnaJ and grpE chaperone gene functions. CONCLUSIONS: The lambda O-containing replication complex is inherited together with DNA and bears information how to initiate the next round of replication at ori lambda; thus, we consider that this phenomenon deserves to be called protein inheritance.

Bacterial Proteins↗
PubMed2006

Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans.

Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukaryotic genomes are among the current challenges in molecular genetics. Variation in gene copy number is increasingly recognized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation. Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glomerulonephritis in Wistar Kyoto rats. In humans, low copy number of FCGR3B, an orthologue of rat Fcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease.

Animals↗
PubMed1994

Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose.

We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.

Acanthamoeba↗
PubMed2002

Retrograde transport of protein toxins under conditions of COPI dysfunction.

Retrograde transport dependent on coat protein I (COPI) was impaired using two different approaches and the effects on the retrograde transport of protein toxins were investigated. One approach was to study ldlF cells that express a temperature-sensitive defect in the epsilon-COP subunit of COPI. The second approach was to treat cells with 1,3-cyclohexanebis(methylamine) (CBM), a drug that interferes with the binding of COPI to Golgi membranes. With both approaches, cells remained sensitive to a variety of protein toxins regardless of whether the toxins contained a KDEL motif. Moreover, cholera toxin, which contains a KDEL sequence, was observed by immunofluorescence microscopy to enter the endoplasmic reticulum of Vero cells in the presence of CBM. These data support published evidence indicating the presence in cells of a COPI- and KDEL receptor-independent pathway of retrograde transport from the Golgi complex to the endoplasmic reticulum. In addition, the results suggest that certain toxins containing a KDEL motif may use either the COPI-dependent or COPI-independent pathway of retrograde transport.

Adaptor Protein Complex gamma Subunits↗
PubMed2001

Four-way junctions in antisense RNA-mRNA complexes involved in plasmid replication control: a common theme?

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. In plasmid R1, we have recently shown that the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by the formation of two intermolecular helices, resulting in a four-way junction structure and a side-by-side helical alignment. Based on lead-induced cleavage and ribonuclease (RNase) V(1) probing combined with molecular modeling, a strikingly similar topology is supported for the complex formed between the antisense RNA (Inc) and mRNA (RepZ) of plasmid Col1b-P9. In particular, the position of the four-way junction and the location of divalent ion-binding site(s) indicate that the structural features of these two complexes are essentially the same in spite of sequence differences. Comparisons of several target and antisense RNAs in other plasmids further indicate that similar binding pathways are used to form the inhibitory antisense-target RNA complexes. Thus, in all these systems, the structural features of both antisense and target RNAs determine the topologically possible and kinetically favored pathway that is essential for efficient in vivo control.

Base Sequence↗
PubMed2001

Demonstration of conformational changes associated with activation of the maltose transport complex.

In Escherichia coli, interaction of a periplasmic maltose-binding protein with a membrane-associated ATP-binding cassette transporter stimulates ATP hydrolysis, resulting in translocation of maltose into the cell. The maltose transporter contains two transmembrane subunits, MalF and MalG, and two copies of a nucleotide-hydrolyzing subunit, MalK. Mutant transport complexes that function in the absence of binding protein are thought to be stabilized in an ATPase-active conformation. To probe the conformation of the nucleotide-binding site and to gain an understanding of the nature of the conformational changes that lead to activation, cysteine 40 within the Walker A motif of the MalK subunit was modified by the fluorophore 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. Fluorescence differences indicated that residues involved in nucleotide binding were less accessible to aqueous solvent in the binding protein independent transporter than in the wild-type transporter. Similar differences in fluorescence were seen when a vanadate-trapped transition state conformation was compared with the ground state in the wild-type transporter. Our results and recent crystal structures are consistent with a model in which activation of ATPase activity is associated with conformational changes that bring the two MalK subunits closer together, completing the nucleotide-binding sites and burying ATP in the interface.

ATP-Binding Cassette Transporters↗
PubMed1998

Prader-Willi and Angelman syndromes. Disorders of genomic imprinting.

Prader-Willi and Angelman syndromes are 2 clinically distinct disorders associated with multiple anomalies and mental retardation. They are only discussed together because they share a similar and uncommon genetic basis: they involve genes that are located in the same region in the genome and are characterized by genetic imprinting. This normal process has contributed to these 2 complex and severe conditions through inactivation of 1 copy of the genes relevant to each disorder: the maternally derived copy of genes for Prader-Willi syndrome in proximal 15q are normally silent, and a paternally derived copy of 1 gene for Angelman syndrome in 15q is normally silent. For both disorders, when the normally active copy of the gene or genes is missing, abnormality results. Since the genes for these 2 disorders are located very close together, and since the center involved in inactivating the genes involved in imprinting may be the same, both these disorders usually result from the same chromosomal deletion; which disorder results depends on the parent of origin of the chromosome 15 that becomes deleted. Both Prader-Willi and Angelman syndrome can also occur as a result of having both members of the chromosome 15 pair derived from 1 parent, a condition known as uniparental disomy. Both can also result from a structural abnormality of the imprinting center, known as an imprinting mutation. In addition, Angelman syndrome can be caused by a mutation in the gene that causes it; a comparable cause is not present in Prader-Willi syndrome since it results from abnormality in more than 1 gene. Finally, despite the complexity of possible causes, all but the single gene mutation of the Angelman syndrome gene can be detected through methylation-sensitive DNA probes, since DNA methylation is the process by which the genes for these 2 disorders are imprinted. This unusual property of specific areas of the DNA holds promise for future treatment of these and other disorders related to imprinting through reversal of the imprinting process.

Adult↗
PubMed1996

Entrapment by immobilized metal ion affinity chromatography of assembled yeast mitochondrial ATP synthase containing individual subunits tagged with hexahistidine.

We demonstrate the use of immobilized metal ion affinity chromatography for isolating the constituent subunits of assembled mitochondrial ATP synthase (mtATPase) wherein a single subunit of the complex has been modified to contain hexahistidine. Genes encoding subunit d or OSCP of mtATPase from Saccharomyces cerevisiae were modified each to encode a polypeptide having a C-terminal addition of six consecutive histidines. Expression of plasmid-borne modified genes, in host yeast cells lacking a functional copy of the relevant endogenous gene, generated functional mtATPase complexes as judged by growth of rescued cells on the nonfermentable substrate ethanol. Significantly, the oligomycin-sensitive ATP hydrolase activity in mitochondria from cells expressing tagged subunits was similar to that of cells expressing unmodified subunits, indicating that there had been no impairment of the functional integrity of mtATPase. Mitochondrial lysates were prepared from each strain and subjected to chromatography under nondenaturing conditions on a resin containing immobilized Ni2+. It is likely that the mtATPase complexes adsorbed by immobilized metal ion affinity chromatography are fully assembled because their subunit composition closely matches that of a preparation of assembled mtATPase conventionally isolated from mitochondrial lysates by ammonium sulfate precipitation and purification by sucrose gradient centrifugation. Furthermore, assembled mtATPase containing a tagged subunit could be adsorbed, albeit at lower yield, when the relevant modified gene was expressed in wild-type host cells. The general application of this novel isolation procedure greatly simplifies and reduces the number of steps required for the isolation of assembled multi-subunit complexes. Moreover, the approach may be used for studying subunit-subunit interactions within the mtATPase complex.

Adenosine Triphosphatases↗
PubMed2001

Reafferent copies of imitated actions in the right superior temporal cortex.

Imitation is a complex phenomenon, the neural mechanisms of which are still largely unknown. When individuals imitate an action that already is present in their motor repertoire, a mechanism matching the observed action onto an internal motor representation of that action should suffice for the purpose. When one has to copy a new action, however, or to adjust an action present in one's motor repertoire to a different observed action, an additional mechanism is needed that allows the observer to compare the action made by another individual with the sensory consequences of the same action made by himself. Previous experiments have shown that a mechanism that directly matches observed actions on their motor counterparts exists in the premotor cortex of monkeys and humans. Here we report the results of functional magnetic resonance experiments, suggesting that in the superior temporal sulcus, a higher order visual region, there is a sector that becomes active both during hand action observation and during imitation even in the absence of direct vision of the imitator's hand. The motor-related activity is greater during imitation than during control motor tasks. This newly identified region has all the requisites for being the region at which the observed actions, and the reafferent motor-related copies of actions made by the imitator, interact.

Adult↗
PubMed1983

Complex-irreversibility and evolution.

Both, irreversibility and evolution, imply order in time. It is argued that the only possible concept of time is a 'system-specific time', and that order in time is convertible into order in space and vice-versa. While life-less, complex systems are irreversible because of their complexity and, hence, not repeatable, living systems are reproduced by irreversible copy-reproduction and by coding. This mode of reproduction results of necessity in an arrow of time of growth and increasing complexity with death as its antagonist, and in obligatory spatial asymmetry. This arrow of increasing organic complexity is simultaneous with, and independent of, the arrow of increasing entropy. - A generalized, organic hierarchy is proposed as the model to study higher evolution. This hierarchy reproduces itself by differential rates of reproduction of its subunits within and between the various hierarchical levels of organization. Phylogenetic change is brought about by a change in this hierarchy's specific phase pattern of growth. Continuous and discrete organization is defined, and it is shown that specific relations between continuous and discrete levels within the hierarchy result in accumulation of neutral alleles. This accumulation is due to complex-irreversibility and causes genetic stabilisation, i.e. heritability, of the species-specific morphology of organisms.

Animals↗
PubMed1992

Variable severity of pulmonary disease in adults with identical cystic fibrosis mutations.

Adults with CF followed in a university center were assessed for the presence of the most common CF gene mutation, delta-F508. Excluding one member of a sibling pair, 29 of 55 subjects had two copies of delta-F508 (homozygotes), 23 had one copy of delta-F508 with the other CF mutation not identified (complex heterozygotes) and three were lacking delta-F508. A wide range of clinical severity was seen among individuals carrying two copies of the delta-F508 gene, who are genetically identical at the CF gene locus. The number of individuals diagnosed with CF as adults was significantly lower in the homozygote group (1 of 29) as compared with the heterozygote group (7 of 24). No differences were detected between groups in pulmonary function, non-pulmonary complications or overall clinical severity. These results suggest that environmental or background genetic factors contribute significantly to the variability in pulmonary and other complications seen among individuals with CF.

Adolescent↗