SEARCH · PubMed Health

Results for “Multiprotein Complexes”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources

PubMed2001

The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs.

How do cells discriminate between selectively edited mRNAs that encode new protein isoforms, and dsRNA-induced, promiscuously edited RNAs that encode nonfunctional, mutant proteins? We have developed a Xenopus oocyte model system which shows that a variety of hyperedited, inosine-containing RNAs are specifically retained in the nucleus. To uncover the mechanism of inosine-induced retention, HeLa cell nuclear extracts were used to isolate a multiprotein complex that binds specifically and cooperatively to inosine-containing RNAs. This complex contains the inosine-specific RNA binding protein p54(nrb), the splicing factor PSF, and the inner nuclear matrix structural protein matrin 3. We provide evidence that one function of the complex identified here is to anchor hyperedited RNAs to the nuclear matrix, while allowing selectively edited mRNAs to be exported.

Active Transport, Cell Nucleus↗

PubMed2006

Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex.

The vaccinia virus G9R gene (VACWR087) encodes a protein of 340 amino acids with the following structural features that are conserved in all poxviruses: a site for N-terminal myristoylation, 14 cysteines, and a C-terminal transmembrane domain. Previous studies showed that G9 is one of eight proteins associated in a putative entry-fusion complex. Our attempt to isolate a mutant without the G9R gene was unsuccessful, suggesting that it is essential for virus replication. To further investigate its role, we constructed a recombinant vaccinia virus in which G9R is regulated by addition of an inducer. Induced G9 protein was associated with mature infectious virions and could be labeled with a membrane-impermeant biotinylation reagent, indicating surface exposure. Omission of inducer reduced the infectious-virus yield by about 1.5 logs; nevertheless, all stages of virus morphogenesis appeared normal and extracellular virions were present on the cell surface. Purified virions assembled without inducer had a specific infectivity of less than 5% of the normal level and a comparably small amount of G9, whereas their overall polypeptide composition, including other components of the entry-fusion complex, was similar to that of virions made in the presence of inducer or of wild-type virions. G9-deficient virions bound to cells, but penetration of cores into the cytoplasm and early viral RNA synthesis were barely detected, and cell-cell fusion was not triggered by low pH. Of the identified components of the multiprotein complex, G9 is the sixth that has been shown to be required for entry and membrane fusion.

Amino Acid Sequence↗

PubMed1990

Mitochondrial rhodanese: membrane-bound and complexed activity.

We have proposed that phosphorylated and dephosphorylated forms of the mitochondrial sulfurtransferase, rhodanese, function as converter enzymes that interact with membrane-bound iron-sulfur centers of the electron transport chain to modulate the rate of mitochondrial respiration (Ogata, K., Dai, X., and Volini, M. (1989) J. Biol. Chem. 204, 2718-2725). In the present studies, we have explored some structural aspects of the mitochondrial rhodanese system. By sequential extraction of lysed mitochondria with phosphate buffer and phosphate buffer containing 20 mM cholate, we have shown that 30% of the rhodanese activity of bovine liver is membrane-bound. Resolution of cholate extracts on Sephadex G-100 indicates that part of the bound rhodanese is complexed with other mitochondrial proteins. Tests with the complex show that it forms iron-sulfur centers when incubated with the rhodanese sulfur-donor substrate thiosulfate, iron ions, and a reducing agent. Experiments on the rhodanese activity of rat liver mitochondria give similar results. Taken together, the findings indicate that liver rhodanese is in part bound to the mitochondrial membrane as a component of a multiprotein complex that forms iron-sulfur centers. The findings are consistent with the role we propose for rhodanese in the modulation of mitochondrial respiratory activity.

Animals↗

PubMed2000

NHERF associations with sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3.

The sodium-hydrogen exchanger regulatory factor (NHERF) is an essential cofactor for cAMP-mediated inhibition of the Na(+)/H(+) exchanger isoform, NHE3, in renal brush border membranes. NHERF is also an ezrin-binding protein. To define the functional importance of ezrin binding for NHERF's function as a NHE3 regulator, we transfected stable PS120 cells expressing NHE3 with plasmids encoding WT and truncated mouse NHERF proteins. Co-immunoprecipitation established that in PS120 cells, NHE3 bound to full-length NHERF(1-355), the C-terminal domain, NHERF(147-355), and NHERF(1-325), which lacks the proposed ezrin-binding domain. The N-terminal domain, NHERF(1-146), failed to bind the antiporter. Ezrin was also co-immunoprecipitated with NHERF(1-355) but not with NHERF(1-325). 8Br-cAMP inhibited NHE3 activity in cells that expressed NHERF(1-355) or NHERF(147-355) but had no effect on the formation of NHE3-NHERF or NHERF-ezrin complexes. Na(+)/H(+) exchange was unaffected by 8Br-cAMP in cells that expressed NHERF(1-146) or NHERF(1-325). NHE3 phosphorylation in vivo was enhanced by 8Br-cAMP only in cells where NHERF bound to both NHE3 and ezrin. The data suggest that NHERF functions as a scaffold to link NHE3 with ezrin and that this multiprotein complex is essential for cAMP-mediated phosphorylation of NHE3 and the inhibition of Na(+)/H(+) exchange.

Animals↗

PubMed2002

Mutagenesis of a functional chimeric gene in yeast identifies mutations in the simian virus 40 large T antigen J domain.

Simian virus 40 large T antigen contains an amino terminal J domain that catalyzes T antigen-mediated viral DNA replication and cellular transformation. To dissect the role of the J domain in these processes, we exploited the genetic tools available only in the yeast Saccharomyces cerevisiae to isolate 14 loss-of-function point mutations in the T antigen J domain. This screen also identified mutations that, when engineered into simian virus 40, resulted in T antigen mutants that were defective for the ability to support viral growth, to transform mammalian cells in culture, to dissociate the p130-E2F4 transcription factor complex, and to stimulate ATP hydrolysis by hsc70, a hallmark of J domain-containing molecular chaperones. These data correlate the chaperone activity of the T antigen J domain with its roles in viral infection and cellular transformation and support a model by which the viral J domain recruits the cytoplasmic hsc70 molecular chaperone in the host to rearrange multiprotein complexes implicated in replication and transformation. More generally, this study presents the use of a yeast screen to identify loss-of-function mutations in a mammalian virus and can serve as a widely applicable method to uncover domain functions of mammalian proteins for which there are yeast homologues with selectable mutant phenotypes.

Adenosine Triphosphate↗

PubMed2006

High throughput protein expression screening in the nervous system--needs and limitations.

The cellular complexity of the brain (some estimate that there are up to 10(3) different cell types) is exceeded by the synaptic complexity, with each of the approximately 10(11) neurons in the brain having around 10(3)-10(4) synapses. Proteomic studies of the synapse have revealed that the postsynaptic density is the most complex multiprotein structure yet identified, with approximately 10(3) different proteins. Such studies, however, use brain tissue with many different regions and therefore different cell types, and there is clear potential for heterogeneity of protein content at different synapses within and between brain regions. Although large-scale mRNA-based assays are in progress to map this sort of complexity at the cellular level, and indeed all brain-expressed genes, analysis of protein distribution (at synapses and other structures) is still in the very early stages. We review existing large-scale protein expression studies and the specific technical obstacles that need to be overcome before applying the scaling used in nucleic acid based approaches.

Animals↗

PubMed2003

The WD motif-containing protein RACK-1 functions as a scaffold protein within the type I IFN receptor-signaling complex.

The WD repeat-containing protein receptor for activated protein kinase C (RACK)-1 has been linked to a variety of signaling systems including protein kinase C, growth factors, and IFNs. In the IFN system, RACK-1 functions as an adaptor recruiting the transcription factor STAT1 to the receptor complex. However, RACK-1 should play a broader role in type I IFN signaling because mutation of the RACK-1 binding site in the IFN-alpha receptor 2/beta subunit of the type I IFN receptor abrogates not only STAT1, but also STAT2, activation. In this study, we demonstrate that RACK-1 serves as a scaffold protein for a multiprotein complex that includes the IFN-alpha receptor 2/beta-chain of the receptor, STAT1, Janus kinase 1, and tyrosine kinase 2. In vitro data further suggest that within this complex tyrosine kinase 2 is the tyrosine kinase responsible for the phosphorylation of STAT1. Finally, we provide evidence that RACK-1 may also serve as a scaffold protein in other cytokine systems such as IL-2, IL-4, and erythropoietin.

Amino Acid Motifs↗

PubMed1988

Analysis of sequential steps of nucleotide excision repair in Escherichia coli using synthetic substrates containing single psoralen adducts.

Escherichia coli ABC excinuclease initiates the removal of dodecanucleotides from damaged DNA in an ATP-dependent reaction. Using a synthetic DNA fragment containing a psoralen adduct at a defined position we have investigated the interaction of the components of the enzyme with substrate by DNase I footprinting. We find that the UvrA subunit binds to DNA specifically in the absence of cofactors and that the binding affinity is stimulated about 4-fold by ATP and only marginally inhibited by ADP. The UvrA.DNA complexes formed in the absence of co-factors or in the presence of either ATP or ADP are remarkably similar. In contrast, adenosine 5'-O-(thiotriphosphate) increases nonspecific binding and completely abolishes the UvrA footprint. The UvrB subunit can associate with the UvrA subunit on DNA in the absence of ATP, but this ternary UvrA.UvrB.DNA complex is qualitatively different from that formed in the presence of ATP. The UvrC subunit elicits no additional change in the UvrA-UvrB footprint. Helicase II (UvrD protein) does not alter the UvrA-UvrB footprint but does appear to interact at the 5'-incision site of the postincision complex. DNA polymerase I fills in the excision gap in the presence or absence of helicase II and apparently releases the ABC excinuclease from the repaired DNA. Nearly 90% of the repair patches are 12 nucleotides long, and this length is not affected by helicase II. We see no evidence by DNase I footprinting for the formation of a multiprotein complex encompassing the UvrA, -B, -C, and -D proteins and DNA polymerase I.

Adenosine Diphosphate↗

PubMed1993

Laser cross-linking of proteins to nucleic acids. I. Examining physical parameters of protein-nucleic acid complexes.

Pulsed laser cross-linking results in efficient and rapid formation of covalent bonds between proteins and the nucleic acids to which they are bound, creating a "snapshot" of the protein-nucleic acid equilibrium existing at the moment of irradiation. The "frozen" equilibrium allows the determination of protein-nucleic acid binding constants, confirming both theoretical predictions and experimental determinations by standard physical chemical methods. Laser cross-linking results accurately reflect the alteration of protein-nucleic acid interactions induced by traditional methods such as increasing the salt concentration or by the addition of a nucleic acid that competes for binding of the protein. Thus this technique is very useful for the study of the association of proteins and protein complexes with nucleic acids under environmental conditions at which the reactions are not amenable to study by traditional physical chemical methods. In this paper we continue our calibration of the method, focusing primarily on interactions with single-stranded DNA-binding proteins and describe techniques for measuring quantitative interactions between nucleic acid constructs and single-protein or multiprotein complexes. Laser cross-linking can also provide direct evidence that binding correlates with functional activity.

Bacterial Proteins↗

PubMed2002

Cell cycle-dependent assembly of a Gin4-septin complex.

Gin4, a Nim1-related kinase, is required in budding yeast for localization of the septins and for proper control of daughter cell growth during G2/M. Gin4 becomes hyperphosphorylated when cells enter mitosis, leading to activation of Gin4 kinase activity. In this study, we have used immunoaffinity chromatography to identify proteins that associate with Gin4 during mitosis, with the goal of finding targets of Gin4 kinase activity and proteins that play a role in Gin4 activation. We show that during mitosis Gin4 is assembled into a multiprotein complex that includes Nap1, Bni5, the septins, and at least two molecules of Gin4. The associated Gin4 molecules present in this complex phosphorylate each other, leading to Gin4 hyperphosphorylation. Furthermore, the Shs1 septin present in the complex undergoes Gin4-dependent phosphorylation during mitosis and appears to be a substrate of Gin4 in vitro, suggesting that it is a target of Gin4 kinase activity in vivo. Genetic data support the idea that Shs1 is an important target of Gin4 kinase activity. Association of Gin4 with the septins during mitosis requires Shs1, Nap1, Cla4, Elm1, and the kinase activities of Gin4 and Cdc28. Self-association of Gin4 molecules requires Shs1 but not Cla4 or Nap1. Previous work has suggested that the septins function together as a tight complex, and we found that the majority of the Shs1 in the cell is tightly bound to the other septins Cdc3, Cdc10, Cdc11, and Cdc12. Interestingly, however, Shs1 can bind to Gin4 and induce Gin4 oligomerization under conditions in which the Cdc11 septin does not bind to Gin4, suggesting that Shs1 can function independently of the other septins. Taken together, these findings suggest that highly regulated protein-binding events ensure that the Gin4 kinase is activated only during mitosis and only in association with Shs1, a likely in vivo substrate of Gin4. In addition, these results provide clues to how Gin4 may regulate the localization or function of the septins.

Cell Cycle↗

PubMed2005

Noncovalent Shiga-like toxin assemblies: characterization by means of mass spectrometry and tandem mass spectrometry.

Shiga-like toxin 1 (SLTx), produced by enterohemorrhagic strains of Escherichia coli (EHEC), belongs to a family of structurally and functionally related AB(5) protein toxins that are associated with human disease. EHEC infection often gives rise to hemolytic colitis, while toxin-induced kidney damage is one of the major causes of hemolytic uremic syndrome (HUS) and acute renal failure in children. As such, an understanding and analysis of the noncovalent interactions that maintain the quaternary structure of this toxin are fundamentally important since such interactions have significant biochemical and medical implications. This paper reports on the analysis of the noncovalent homopentameric complex of Shiga-like toxin B chain (SLTx-B(5)) using electrospray ionization (ESI) triple-quadrupole (QqQ) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) and the analysis of the noncovalent hexameric holotoxin (SLTx-AB(5)) using ESI time-of-flight (TOF) MS. The triple-quadrupole analysis revealed highly charged monomer ions dissociate from the multiprotein complex to form dimer, trimer, and tetramer product ions, which were also seen to further dissociate. The ESI-TOFMS analysis of SLTx-AB(5) revealed the complex remained intact and was observed in the gas phase over a range of pHs. Theses findings demonstrate that the gas-phase structure observed for both the holotoxin and the isoloated B chains correlates well with the structures reported to exist in the solution phase for these proteins. Such analysis provides a rapid screening technique for assessing the noncovalent structure of this family of proteins and other structurally related toxins.

Hydrogen-Ion Concentration↗

PubMed2004

Multiple TPR motifs characterize the Fanconi anemia FANCG protein.

The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.

Amino Acid Sequence↗

PubMed2000

Mints as adaptors. Direct binding to neurexins and recruitment of munc18.

Mint1 (X11/human Lin-10) and Mint2 are neuronal adaptor proteins that bind to Munc18-1 (n/rb-sec1), a protein essential for synaptic vesicle exocytosis. Mint1 has previously been characterized in a complex with CASK, another adaptor protein that in turn interacts with neurexins. Neurexins are neuron-specific cell surface proteins that act as receptors for the excitatory neurotoxin alpha-latrotoxin. Hence, one possible function for Mint1 is to mediate the recruitment of Munc18 to neurexins. In agreement with this hypothesis, we now show that the cytoplasmic tail of neurexins captures Munc18 via a multiprotein complex that involves Mint1. Furthermore, we demonstrate that both Mint1 and Mint2 can directly bind to neurexins in a PDZ domain-mediated interaction. Various Mint and/or CASK-containing complexes can be assembled on neurexins, and we demonstrate that Mint1 can bind to Munc18 and CASK simultaneously. Our data support a model whereby one of the functions of Mints is to localize the vesicle fusion protein Munc18 to those sites at the plasma membrane that are defined by neurexins, presumably in the vicinity of points of exocytosis.

Adaptor Proteins, Signal Transducing↗

PubMed1998

The 3' to 5' degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3' to 5' exonucleases of the exosome complex.

One major pathway of mRNA decay in yeast occurs by deadenylation-dependent decapping, which exposes the transcript to 5' to 3' exonucleolytic degradation. We show that a second general pathway of mRNA decay in yeast occurs by 3' to 5' degradation of the transcript. We also show that the SKI2, SKI3, SKI6/RRP41, SKI8 and RRP4 gene products are required for 3' to 5' decay of mRNA. The Ski6p/Rrp41p protein has homology to the Escherichia coli 3' to 5' exoribonuclease RNase PH, and both the Ski6p/Rrp41p and Rrp4p proteins are components of a multiprotein complex, termed the exosome, that contains at least three polypeptides with 3' to 5' exoribonuclease activities. These observations suggest that the exosome may be the nucleolytic activity that degrades the body of the mRNA in a 3' to 5' direction, and the exosome's activity on mRNAs may be modulated by Ski2p, Ski3p and Ski8p. Blocking both 3' to 5' and 5' to 3' decay leads to inviability, and conditional double mutants show extremely long mRNA half-lives. These observations argue that efficient mRNA turnover is required for viability and that we have identified the two major pathways of mRNA decay in yeast.

Endoribonucleases↗

PubMed2004

The Shp-1 and Shp-2, tyrosine phosphatases, are recruited on cell membrane in two distinct molecular complexes including Ret oncogenes.

The Shp-2 and Shp-1 non-transmembrane tyrosine phosphatases display different and even opposing effects on downstream signaling events initiated by Ret activation. By using rat pheochromocytoma-derived PC12 cells, here we studied the interactions of Shp-2 and Shp-1 with two activated mutants of Ret receptor, Ret(C634Y) and Ret(M918T). Each of these mutated receptors causes inheritance of distinct cancer syndromes, multiple endocrine neoplasia (MEN) type 2A and type 2B, respectively. We show that: (i) both Shp-1 and Shp-2 are associated to a multiprotein complex that includes Ret mutants; (ii) the Shp-1-Ret complexes are distinct from Shp-2-Ret complexes, and these complexes are differently distributed inside and outside lipid rafts; (iii) constitutively activated Ret proteins neither directly bind to nor are substrates of these phosphatases. Our results well support the evidence that Ret complexes within and outside rafts mediate distinct biological functions, and indicate that the presence of either Shps participates to determine such functions.

Animals↗

PubMed1969

Nonsense mutations affecting the his4 enzyme complex of yeast.

The his4 region of yeast contains the information necessary for the catalysis of three steps in the histidine biosynthetic pathway. The three activities specified by this region remain physically associated during gel filtration and ultracentrifugation and after extensive purification. Nonsense mutations in the "operator distal" his4C region lower the molecular weight of the two proximal activities. The proximal activities can, therefore, function without the his4C portion of the protein(s). These observations are compatible with the following three possibilities. The his4 region codes for: (1) an aggregate of three proteins forming a multiprotein complex; (2) a single protein with three catalytic activities; and (3) a single protein which is cleaved to form three proteins which remain associated.

Chromatography, Gel↗

PubMed2003

Novel Drosophila heterochromatin protein 1 (HP1)/origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations.

Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin.

Amino Acid Motifs↗

PubMed1993

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase alpha complex.

We have analyzed the ATPase and dATPase activities associated with the yeast DNA polymerase alpha complex. The ATPase/dATPase was primarily a single-stranded DNA-dependent ATPase. Analysis of the stimulatory effect of a large number of DNA substrates demonstrated that polynucleotides longer than 60 nucleotides (nts) had the maximal effect. The stimulation by oligonucleotides smaller than 60 nts, in general, decreased proportionally with decreased length of the oligomer. Poly- or oligopyrimidines were twice as stimulatory as the poly- or oligopurines of the same length. In addition to DNA, replication protein A (RP-A), a single-stranded DNA (ssDNA) binding protein, also stimulated the ATPase activity. Photo-cross-linking of the ATP binding component of the pol alpha complex to [alpha-32P]ATP at 0 degree C resulted in the exclusive labeling of a 90-kDa polypeptide. The labeling was inhibited by ATP and dATP but not by any other ribo- or deoxynucleotides, which suggest that the 90-kDa polypeptide is specific for ATP/dATP binding and possibly the active site for the ATPase/dATPase. We have also reported here a novel DNA unwinding activity associated with the multiprotein complex of DNA polymerase alpha. The complex was able to unwind M13mp19 ssDNA hybridized to an oligonucleotide (17-60 nucleotides long) with a protruding 3'-terminus. Regardless of the size of the duplex, the DNA unwinding was significantly stimulated by RP-A, while RP-A itself did not have any DNA unwinding activity. Consequently, it appeared that the DNA polymerase alpha complex possessed a putative RP-A-dependent helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗