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PubMed1997

Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits. Possible roles in RNA binding and macromolecular assembly.

The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.

Amino Acid Sequence↗

PubMed1997

Analysis of chromatin structure by in vivo formaldehyde cross-linking.

Recent advances leave no doubt that higher order chromatin structures play a fundamental role in many developmentally important mechanisms of gene regulation. In particular analyses in genetic model systems like yeast and Drosophila uncovered novel proteins that are involved in the regulation of chromatin structures. Many of these proteins do not bind directly to DNA but interact in large multimeric complexes. To identify the DNA elements regulated by these multiprotein complexes, alternative approaches to the standard methods of DNA-protein analysis had to be devised. Here we present a method that preserves the architecture of the higher order chromatin structures by cross-linking cells in vivo with formaldehyde. An immunoprecipitation strategy is then used to identify the DNA targets of chromosomal proteins of interest. This method can be applied to study the distribution of proteins at high resolution over extended chromosomal regions.

Animals↗

PubMed1995

Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex.

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex (NPC). Therefore, the possibility exists that NPCs restrict TBP translocation to the nuclear interior. Here we show for the first time, with patch-clamp and atomic force microscopy (AFM), that NPCs regulate TBP movement into the nucleus and that TBP (10(-15)-10(-10)M) is capable of modifying NPC structure and function. The translocation of TBP was ATP-dependent and could be detected as a transient plugging of the NPC channels, with a concomitant transient reduction in single NPC channel conductance, gamma, to a negligible value. NPC unplugging was accompanied by permanent channel opening at concentrations greater than 250 pM. AFM images demonstrated that the TBP molecules attached to and accumulated on the NPC cytosolic side. NPC channel activity could be recorded for more than 48 hr. These observations suggest that three novel functions of TBP are: to stabilize NPC, to force the NPC channels into an open state, and to increase the number of functional channels. Since TBP is a major component of transcription, our observations are relevant to the understanding of the gene expression mechanisms underlying normal and pathological cell structure and function.

Animals↗

PubMed1998

Targeted recruitment of the Sin3-Rpd3 histone deacetylase complex generates a highly localized domain of repressed chromatin in vivo.

Eukaryotic organisms contain a multiprotein complex that includes Rpd3 histone deacetylase and the Sin3 corepressor. The Sin3-Rpd3 complex is recruited to promoters by specific DNA-binding proteins, whereupon it represses transcription. By directly analyzing the chromatin structure of a repressed promoter in yeast cells, we demonstrate that transcriptional repression is associated with localized histone deacetylation. Specifically, we observe decreased acetylation of histones H3 and H4 (preferentially lysines 5 and 12) that depends on the DNA-binding repressor (Ume6), Sin3, and Rpd3. Mapping experiments indicate that the domain of histone deacetylation is highly localized, occurring over a range of one to two nucleosomes. Taken together with previous observations, these results define a novel mechanism of transcriptional repression which involves targeted recruitment of a histone-modifying activity and localized perturbation of chromatin structure.

Chromatin↗

PubMed1997

Gcn5p is involved in the acetylation of histone H3 in nucleosomes.

Enzymatic extracts from a gcn5 mutant and wild-type strains of Saccharomyces cerevisiae were chromatographically fractionated and the histone acetyltransferase activities compared. When free histones were used as substrate, extracts from wild-type cells showed two peaks of activity on histone H3 but extracts from gcn5 mutant cells showed only one. With nucleosomes as substrate, the histone acetyltransferase activities present in extracts from the gcn5 mutant strain were not able to modify H3 whereas wild-type cell extracts acetylated intensely this histone. The activity that acetylated nucleosome-bound H3 behaved as a 170-kDa complex. We suggest that Gcn5p represents a catalytic subunit within a multiprotein complex containing proteins that confer on it the ability to acetylate H3 in nucleosomes.

Acetylation↗

PubMed1997

Structure of ADP x AIF4(-)-stabilized nitrogenase complex and its implications for signal transduction.

The coupling of ATP hydrolysis to electron transfer by the enzyme nitrogenase during biological nitrogen fixation is an important example of a nucleotide-dependent transduction mechanism. The crystal structure has been determined for the complex between the Fe-protein and MoFe-protein components of nitrogenase stabilized by ADP x AIF4-, previously used as a nucleoside triphosphate analogue in nucleotide-switch proteins. The structure reveals that the dimeric Fe-protein has undergone substantial conformational changes. The beta-phosphate and AIF4- groups are stabilized through intersubunit contacts that are critical for catalysis and the redox centre is repositioned to facilitate electron transfer. Interactions in the nitrogenase complex have broad implications for signal and energy transduction mechanisms in multiprotein complexes.

Adenosine Diphosphate↗

PubMed1997

Functional complementation of an oxa1- yeast mutation identifies an Arabidopsis thaliana cDNA involved in the assembly of respiratory complexes.

The nuclear gene OXA1 is essential for respiratory growth in yeast. It codes for a chaperon-like protein, and has pleiotropic effects on the assembly of cytochrome c oxidase and ATP synthase of the mitochondrial respiratory chain. To study respiratory complex formation in plants, we have cloned a homolog of the yeast oxa1- in Arabidopsis thaliana, OXA1At, by functional complementation of a yeast oxa1- mutant. OXA1At is a single copy gene and appears to be constitutively expressed in A. thaliana. Although OXA1At encodes a protein sharing only 30% amino acid identity with the yeast Oxa1 protein, hydrophobic domains likely corresponding to trans-membrane domains are strictly conserved. Cytochrome spectra and measurements of respiratory activities show that replacement of the yeast Oxa1 protein with the A. thaliana homolog leads to correct assembly and activity of cytochrome c oxidase, but to partial restoration of ATPase activity. Our results suggest that the Oxa1At protein is essential for the respiratory complex assembly in A. thaliana, and that genes involved in mitochondrial multiprotein complex formation can be conserved between plants and other organisms.

Amino Acid Sequence↗

PubMed1997

Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds.

The human aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator protein (ARNT) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of this heterodimeric transcription factor. Specific transcriptional activation mediated by AHR/ARNT heterodimer, which is a functional indicator of receptor expression, was assessed by beta-galactosidase activity produced from a reporter plasmid. Yeast expressing AHR and ARNT displayed constitutive transcriptional activity that was not augmented by addition of AHR agonists in strains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway for tryptophan biosynthesis responded to AHR agonists and had lower levels of background beta-galactosidase activity. Hexachlorobenzene, benzo(a)pyrene, and beta-naphthoflavone were effective AHR agonists in the yeast system, and had EC50 values of 200, 40, and 20 nM, respectively, for beta-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamine activated transcription in yeast coexpressing AHR and ARNT (EC50 values approximately 300 microM). Indole-3-carbinol was an exceptionally potent AHR agonist (EC50 approximately 10 microM) in yeast. This yeast system is useful for the study of AHR/ARNT protein complexes, and may be generally applicable to the investigation of other multiprotein complexes.

Antioxidants↗

PubMed1999

Chromatin and transcription in Saccharomyces cerevisiae.

A central problem in eukaryotic transcription is how proteins gain access to DNA packaged in nucleosomes. Research on the interplay between chromatin and transcription has progressed with the use of yeast genetics as a useful tool to characterize factors involved in this process. These factors have both positive and negative effects on the stability of nucleosomes, thereby controlling the role of chromatin in transcription in vivo. The negative effectors include the structural components of chromatin, the histones and non-histone chromatin associated proteins, as well as regulatory components like chromatin assembly factors and histone deacetylase complexes. The positive factors are involved in remodeling chromatin and several multiprotein complexes have been described: Swi/Snf, Srb/mediator and SAGA. The components of each of these complexes, as well as the functional relationships between them are covered by this review.

Chromatin↗

PubMed1993

Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

Amino Acid Sequence↗

PubMed1994

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

Amino Acid Sequence↗

PubMed1995

Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination.

Regulation of transcription involves the assembly of multiprotein complexes at enhancers and promoters. Interactions between adjacent and non-adjacent DNA-binding proteins can augment the specificity and stability of multi-component nucleoprotein complexes. Recently, several proteins have been identified that can function as 'architectural' elements in the assembly of higher-order nucleoprotein structures reminiscent of those involved in site-specific recombination in prokaryotes.

Animals↗

PubMed1999

Chromatin-modifying and -remodeling complexes.

Nucleosomes have long been known to inhibit DNA transactions on chromosomes and a remarkable abundance of multiprotein complexes that either enhance or relieve this inhibition have been described. Most is known about chromatin-remodeling complexes that perturb nucleosome structure.

Chromatin↗

PubMed1999

SNARE complex at the ribbon synapses of cochlear hair cells: analysis of synaptic vesicle- and synaptic membrane-associated proteins.

Neurotransmitters are released via exocytosis of synaptic vesicles involving a fusion complex consisting of a set of highly conserved proteins, which form a multiprotein complex resulting in the docking of synaptic vesicles at the site of release. There are three major differences between cochlear hair cell synapses and CNS synapses: (i) hair cells have a specialized structure, the synaptic ribbon, to which synaptic vesicles are attached; (ii) hair cells can maintain high and sustained release of neurotransmitter; and (iii) hair cells lack synaptophysin and synapsin. These differences suggest that an unconventional mechanism of neurotransmitter release may be involved at ribbon synapses. In this study we used different and complementary approaches to determine whether or not ribbon-containing hair cells of the cochlea express any component of the core fusion complex found in conventional synapses. Syntaxin 1, the synaptic membrane synaptosome-associated protein (SNAP)-25 and vesicle-associated membrane protein (VAMP or synaptobrevin) were found to be present in the organ of Corti of both rat and guinea-pig, as shown by reverse transcription polymerase chain reaction and Western blotting. In situ hybridization and immunocytochemistry showed mRNA and protein expression, respectively, in both inner and outer hair cells. Synaptotagmins I and II, generally considered to play major roles in neurotransmitter release at central synapses, were not detected in the organ of Corti.

Animals↗

PubMed1996

Nucleotides reveal polynucleotide phosphorylase activity from conventionally purified GroEL.

GroEL, as conventionally purified, can be incubated with nucleotides to produce high molecular weight material with an absorption maximum at 260 nm. This material is most clearly demonstrated when samples are subjected to gel filtration under conditions where GroEL is monomeric. There is a time-dependent increase in the high molecular weight material that occurs on incubation with ADP or, more slowly, with ATP. This material is generated during incubation, and none is present in the initial samples. Experiments with nucleases, proteases, radiolabeled nucleotides, and chemical cleavage reagents demonstrate that the high molecular weight material is polyadenylic acid whose formation is inhibited by phosphate. These results are consistent with the GroEL samples containing polynucleotide phosphorylase activity. Nondenaturing gels stained with acridine orange, after incubation in ADP, reveal that the activity producing the poly(A) coelectrophoreses with authentic polynucleotide phosphorylase. Conditions that remove the tryptophan-like fluorescence from preparations of GroEL also remove the PNPase activity. Thus, this activity is not associated with GroEL itself. The results are consistent with reports that GroEL can associate with RNase E and with other studies showing that RNase E and PNPase can form complexes. Thus, the present experiments support suggestions that GroEL can participate in multiprotein complexes that are involved in mRNA processing and degradation.

Adenosine Diphosphate↗

PubMed1995

Chromatin-mediated transcriptional repression in yeast.

Chromatin is linked to two modes of transcriptional repression in yeast: silencing at the HM loci and telomeres, and repression by the global regulator complex Ssn6/Tup1. In both cases, unique chromatin configurations appear to be orchestrated by multiprotein complexes targeted to particular regions through interactions with specific DNA-binding proteins, such as RAP1, the origin-recognition complex, and alpha 2.

Chromatin↗

PubMed1992

Cyclins A and B1 in the human cell cycle.

Cyclins are a family of proteins involved in the regulation of the eukaryotic cell cycle. The first cyclins to be isolated were the A- and B-type cyclins and we have been studying their behaviour in human somatic cells. The levels of both cyclin A and B1 are regulated transcriptionally as well as post-translationally; both are rapidly and specifically degraded in mitosis. Cyclin A synthesis commences at the start of S phase and the protein is predominantly nuclear, whereas cyclin B1 appears during S phase and is primarily cytoplasmic. Cyclin B1 moves into the nucleus just at the start of mitosis and associates with condensed chromosomes and the mitotic spindle. Both cyclin A and cyclin B1 bind to and activate a protein serine/threonine kinase subunit; cyclin A associates with p33cdk2 and with p34cdc2, whereas cyclin B1 seems to bind exclusively to p34cdc2. Cyclin A-associated kinase activity appears much earlier in the cell cycle than that of cyclin B1, which appears only at the G2 to M transition. Therefore cyclin A may play a role in the events of S phase as well as G2 and M phases. Cyclin A forms a cell cycle-dependent complex with p33cdk2 and the transcription factor E2F, although the function of this complex is not yet clear. We conclude that cyclins A and B1 may differentially regulate the cell cycle in several ways. They form complexes with distinct protein kinases and these complexes are active at different times in the cell cycle; they form distinct multiprotein complexes, such as with the transcription factor E2F; and they are localized to different parts of the cell where different substrates will be available to them.

Amino Acid Sequence↗

PubMed1997

Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin.

Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506- or cyclosporin A-binding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315-32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormone-free cells. Approximately one-half of the GR.hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and approximately 35% contains PP5. Only a small (but undetermined) fraction of the native GR.hsp90 heterocomplexes contain the cyclosporin A-binding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [3H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol.

Animals↗