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PubMed2005

Structural and functional characterization of PC2 and RNA polymerase II-associated subpopulations of metazoan Mediator.

The coactivator complexes TRAP/SMCC and PC2 represent two forms of Mediator. To further understand the implications of the heterogeneity of the cellular Mediator populations for regulation of RNA polymerase II (Pol II) transcription, we used a combination of affinity and conventional chromatographic methods. Our analysis revealed a spectrum of complexes, including some containing significant proportions of Pol II. Interestingly, the subunit composition of the Pol II-associated Mediator population resembled that of PC2 more closely than that of the larger TRAP/SMCC complex. In in vitro transcription assays reconstituted from homogeneous preparations of general transcription factors, Mediator-associated Pol II displayed a greater specific activity (relative to that of standard Pol II) in activator-independent (basal) transcription in addition to the previously described effects of Mediator on activator-dependent transcription. Purified PC2 complex also stimulated basal activity under these conditions. Immobilized template assays in which activator-recruited preinitiation complexes were allowed to undergo one cycle of transcription revealed partial disruption of Mediator that resulted in a PC2-like complex being retained in the scaffold. This result implies that PC2 could originate as a result of a normal cellular process. Our results are thus consistent with a dynamic nature of the Mediator complex and further extend the functional similarities between Saccharomyces cerevisiae and metazoan Mediator complexes.

HeLa Cells↗
PubMed1993

The glucocorticoid receptor precludes the binding of a transcriptional repressor protein to the long terminal repeat of the mouse mammary tumor virus.

The long terminal repeat (LTR) of the mouse mammary tumor virus was used as a template to examine the dual binding parameters of the glucocorticoid-receptor (GR) and a repressor protein termed Inhibitory Factor 1 (IF1). The receptor binds specifically to the glucocorticoid response element and precludes the binding of IF1 to its juxtaposed binding site within the LTR. When the two DNA targets are separated by the insertion of an additional 52 base pairs, coincident binding of both proteins is observed. Gel retention assays reveal three distinct nucleoprotein complexes. The first complex consists of the receptor and the LTR, the second is comprised of IF1 and DNA and the third is a multiprotein-DNA complex consisting of the GR, IF1 and DNA, migrating at a higher molecular weight position. The inhibition of IF1 binding by the presence of prebound GR leads to the repression of transcription of juxtaposed genes. The GR may act to block access of a sequence, used by the cell to titrate repressor proteins and facilitate the onset of gene expression.

Animals↗
PubMed2003

Fission yeast COP9/signalosome suppresses cullin activity through recruitment of the deubiquitylating enzyme Ubp12p.

The COP9/signalosome (CSN) is known to remove the stimulatory NEDD8 modification from cullins. The activity of the fission yeast cullins Pcu1p and Pcu3p is dramatically stimulated when retrieved from csn mutants but inhibited by purified CSN. This inhibition is independent of cullin deneddylation but mediated by the CSN-associated deubiquitylating enzyme Ubp12p, which forms a complex with Pcu3p in a CSN-dependent manner. In ubp12 mutants, as in csn mutants, Pcu3p activity is stimulated. CSN is required for efficient targeting of Ubp12p to the nucleus, where both cullins reside. Finally, the CSN/Ubp12p pathway maintains the stability of the Pcu1p-associated substrate-specific adaptor protein Pop1p. We propose that CSN/Ubp12p-mediated deubiquitylation creates an environment for the safe de novo assembly of cullin complexes by counteracting the autocatalytic destruction of adaptor proteins.

Active Transport, Cell Nucleus↗
PubMed2005

The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae.

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.

Adenosine Triphosphatases↗
PubMed2004

Crystal structure of a human CD3-epsilon/delta dimer in complex with a UCHT1 single-chain antibody fragment.

The alpha/beta T cell receptor complex transmits signals from MHC/peptide antigens through a set of constitutively associated signaling molecules, including CD3-epsilon/gamma and CD3-epsilon/delta. We report the crystal structure at 1.9-A resolution of a complex between a human CD3-epsilon/delta ectodomain heterodimer and a single-chain fragment of the UCHT1 antibody. CD3-epsilon/delta and CD3-epsilon/gamma share a conserved interface between the Ig-fold ectodomains, with parallel packing of the two G strands. CD3-delta has a more electronegative surface and a more compact Ig fold than CD3-gamma; thus, the two CD3 heterodimers have distinctly different molecular surfaces. The UCHT1 antibody binds near an acidic region of CD3-epsilon opposite the dimer interface, occluding this region from direct interaction with the TCR. This immunodominant epitope may be a uniquely accessible surface in the TCR/CD3 complex, because there is overlap between the binding site of the UCHT1 and OKT3 antibodies. Determination of the CD3-epsilon/delta structure completes the set of TCR/CD3 globular ectodomains and contributes information about exposed CD3 surfaces.

CD3 Complex↗
PubMed2005

Atomic force microscopy reveals the stoichiometry and subunit arrangement of 5-HT3 receptors.

The 5-HT3 receptor is a cation-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor is an important therapeutic target, with receptor antagonists being widely used as antiemetics in cancer therapy. The two known receptor subunits, A and B, form homomeric 5-HT 3A receptors and heteromeric 5-HT 3A/B receptors. The heteromeric receptor has the higher single-channel conductance and more closely mimics the properties of the native receptor. We have used atomic force microscopy to study the architecture of 5-HT 3A and 5-HT 3A/B receptors. We engineered different epitope tags onto the A- and B-subunits and imaged receptors that were doubly liganded by anti-epitope antibodies. We found that, for the 5-HT 3A/B receptor, the distribution of angles between antibodies against the A-subunit had a single peak at approximately 144 degrees , whereas the distribution for antibodies against the B-subunit had two peaks at approximately 72 degrees and 144 degrees . Our results indicate that the subunit stoichiometry is 2A:3B and that the subunit arrangement around the receptor rosette is B-B-A-B-A. This arrangement may account for the difference between the agonist Hill coefficients and the single-channel conductances for the two types of receptor.

Animals↗
PubMed2001

Saccharomyces cerevisiae protein Pci8p and human protein eIF3e/Int-6 interact with the eIF3 core complex by binding to cognate eIF3b subunits.

Mammalian, plant, and Schizosaccharomyces pombe eukaryotic initiation factor-3 (eIF3) contains a protein homologous to the product of int-6 (eIF3e), a frequent integration site of mouse mammary tumor viruses. By contrast, Saccharomyces cerevisiae does not encode a protein closely related to eIF3e/Int-6. Here, we characterize a novel S. cerevisiae protein (Pci8p, Yil071cp) that contains a PCI (proteasome-COP9 signalosome-eIF3) domain conserved in eIF3e/Int-6. We show that both Pci8p and human eIF3e/Int-6 expressed in budding yeast interact with the yeast eIF3 complex in vivo and in vitro by binding to a discrete segment of its eIF3b subunit Prt1p and that human eIF3e/Int-6 interacts with the human eIF3b segment homologous to the Pci8p-binding site of yeast Prt1p. These results refine our understanding of subunit interactions in the eIF3 complex and suggest structural similarity between human eIF3e/Int-6 and yeast Pci8p. However, deletion of PCI8 had no discernible effect on cell growth or translation initiation as judged by polysome analysis, suggesting that Pci8p is not required for the essential function of eIF3 in translation initiation. Motivated by the involvement of Int-6 in transcriptional control, we investigated the effects of deleting PCI8 on the total mRNA expression profile by oligonucleotide microarray analysis and found reduced mRNA levels for a subset of heat shock proteins in the pci8Delta mutant. We discuss possible dual functions of Pci8p and Int-6 in transcriptional and translational control.

Binding Sites↗
PubMed2006

Amino-terminal dimerization, NRDP1-rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease 8 (USP8).

Ubiquitin-specific protease 8 (USP8) hydrolyzes mono and polyubiquitylated targets such as epidermal growth factor receptors and is involved in clathrin-mediated internalization. In 1182 residues, USP8 contains multiple domains, including coiled-coil, rhodanese, and catalytic domains. We report the first high-resolution crystal structures of these domains and discuss their implications for USP8 function. The amino-terminal domain is a homodimer with a novel fold. It is composed of two five-helix bundles, where the first helices are swapped, and carboxyl-terminal helices are extended in an antiparallel fashion. The structure of the rhodanese domain, determined in complex with the E3 ligase NRDP1, reveals the canonical rhodanese fold but with a distorted primordial active site. The USP8 recognition domain of NRDP1 has a novel protein fold that interacts with a conserved peptide loop of the rhodanese domain. A consensus sequence of this loop is found in other NRDP1 targets, suggesting a common mode of interaction. The structure of the carboxyl-terminal catalytic domain of USP8 exhibits the conserved tripartite architecture but shows unique traits. Notably, the active site, including the ubiquitin binding pocket, is in a closed conformation, incompatible with substrate binding. The presence of a zinc ribbon subdomain near the ubiquitin binding site further suggests a polyubiquitin-specific binding site and a mechanism for substrate induced conformational changes.

Catalytic Domain↗
PubMed2002

Arabidopsis COP10 is a ubiquitin-conjugating enzyme variant that acts together with COP1 and the COP9 signalosome in repressing photomorphogenesis.

A group of evolutionarily conserved pleiotropic COP/DET/FUS proteins was initially defined by their ability to repress photomorphogenesis in Arabidopsis. It was proposed that this regulation be mediated by targeting degradation of key cellular regulators that promote photomorphogenesis. Among them, COP1 and the COP9 signalosome have been hypothesized to fulfill the roles as an ubiquitin ligase (E3) and an essential E3 modulator. Here we report that COP10 encodes a protein similar to ubiquitin-conjugating enzyme (E2) variant proteins (UEV). COP10 is part of a nuclear protein complex and capable of directly interacting with both COP1 and the COP9 signalosome. Our data indicates that COP10 defines a possible E2 activity, thus validating the working hypothesis that the pleiotropic COP/DET/FUS group of proteins defined a protein ubiquitination pathway.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of subunit 6 of the COP9 signalosome and its role in multifaceted developmental processes in Arabidopsis.

The COP9 signalosome is a highly conserved protein complex initially identified as a repressor of photomorphogenesis. Here, we report that subunit 6 of the Arabidopsis COP9 signalosome is encoded by a family of two genes (CSN6A and CSN6B) located on chromosomes V and IV, respectively. The CSN6A and CSN6B proteins share 87% amino acid identity and contain a MPR1p and PAD1p N-terminal (MPN) domain at the N-terminal region. The CSN6 proteins share homology with CSN5 and belong to the Mov34 superfamily of proteins. CSN6 proteins present only in the complex form and coimmunoprecipitate with other known subunits of the COP9 signalosome. Partial loss-of-function strains of the COP9 signalosome created by antisense and cosuppression with CSN6A exhibit diverse developmental defects, including homeotic organ transformation, symmetric body organization, and organ boundary definition. Protein blot analysis revealed that the defective plants accumulate significant amounts of ubiquitinated proteins, supporting the conclusion that the COP9 signalosome regulates multifaceted developmental processes through its involvement in ubiquitin/proteasome-mediated protein degradation.

Amino Acid Sequence↗
PubMed2006

Specific SNARE complex binding mode of the Sec1/Munc-18 protein, Sec1p.

The Sec1/Munc-18 (SM) family of proteins is required for vesicle fusion in eukaryotic cells and has been linked to the membrane-fusion proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SM proteins may activate the target-membrane SNARE, syntaxin, for assembly into the fusogenic SNARE complex. In support of an activation role, SM proteins bind directly to their cognate syntaxins. An exception is the yeast Sec1p, which does not bind the yeast plasma-membrane syntaxin, Sso1p. This exception could be explained if the SM interaction motif were blocked by the highly stable closed conformation of Sso1p. We tested the possibility of a latent binding motif using sso1 mutants in yeast and reconstituted the Sec1p binding specificity observed in vivo with purified proteins in vitro. Our results indicate there is no latent binding motif in Sso1p. Instead, Sec1p binds specifically to the ternary SNARE complex, with no detectable binding to the binary t-SNARE complex or any of the three individual SNAREs in their uncomplexed forms. We propose that vesicle fusion requires a specific interaction between the SM protein and the ternary SNARE complex.

Multiprotein Complexes↗
PubMed2003

Physical and functional modularity of the protein network in yeast.

While protein-protein interactions have been studied largely as a network graph without physicality, here we analyze two protein complex data sets of Saccharomyces cerevisiae to relate physical and functional modularity to the network topology. We study for the first time the number of different protein complexes as a function of the protein complex size and find that it follows an exponential decay with a characteristic number of about 7. This reflects the dynamics of complex formation and dissociation in the cell. The analysis of the protein usage by complexes shows an extensive sharing of subunits that is due to the particular organization of the proteome into physical complexes and functional modules. This promiscuity accounts for the high clustering in the protein net-work graph. Our results underscore the need to include the information contained in observed protein complexes into protein network analyses.

Models, Theoretical↗
PubMed2005

Multi-component protein complexes and Golgi membrane trafficking.

Several large cytosolic protein complexes with multiple components have been proposed to play key roles in mediating or controlling membrane trafficking. Among these complexes, TRAPP, COG and GARP/VFT have been implicated in multiple steps of Golgi membrane trafficking. The importance of these complexes for Golgi function has been established using in vitro biochemical assays and yeast and mammalian somatic cell genetics. Furthermore, mutations in the genes encoding subunits of either TRAPP or the COG complex have been shown to be responsible for human genetic disorders. We here review recent studies exploring the structures and functions of these three oligomeric complexes.

Animals↗
PubMed2006

'Fractional recovery' analysis of a presynaptic synaptotagmin 1-anchored endocytic protein complex.

BACKGROUND: The integral synaptic vesicle protein and putative calcium sensor, synaptotagmin 1 (STG), has also been implicated in synaptic vesicle (SV) recovery. However, proteins with which STG interacts during SV endocytosis remain poorly understood. We have isolated an STG-associated endocytic complex (SAE) from presynaptic nerve terminals and have used a novel fractional recovery (FR) assay based on electrostatic dissociation to identify SAE components and map the complex structure. The location of SAE in the presynaptic terminal was determined by high-resolution quantitative immunocytochemistry at the chick ciliary ganglion giant calyx-type synapse. METHODOLOGY/PRINCIPLE FINDINGS: The first step in FR analysis was to immunoprecipitate (IP) the complex with an antibody against one protein component (the IP-protein). The immobilized complex was then exposed to a high salt (1150 mM) stress-test that caused shedding of co-immunoprecipitated proteins (co-IP-proteins). A Fractional Recovery ratio (FR: recovery after high salt/recovery with control salt as assayed by Western blot) was calculated for each co-IP-protein. These FR values reflect complex structure since an easily dissociated protein, with a low FR value, cannot be intermediary between the IP-protein and a salt-resistant protein. The structure of the complex was mapped and a blueprint generated with a pair of FR analyses generated using two different IP-proteins. The blueprint of SAE contains an AP180/X/STG/stonin 2/intersectin/epsin core (X is unknown and epsin is hypothesized), and an AP2 adaptor, H-/L-clathrin coat and dynamin scission protein perimeter. Quantitative immunocytochemistry (ICA/ICQ method) at an isolated calyx-type presynaptic terminal indicates that this complex is associated with STG at the presynaptic transmitter release face but not with STG on intracellular synaptic vesicles. CONCLUSIONS/SIGNIFICANCE: We hypothesize that the SAE serves as a recognition site and also as a seed complex for clathrin-mediated synaptic vesicle recovery. The combination of FR analysis with quantitative immunocytochemistry provides a novel and effective strategy for the identification and characterization of biologically-relevant multi-molecular complexes.

Animals↗
PubMed2006

Saccharomyces cerevisiae Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation.

In budding yeast, at least 10 proteins are required for formation of the double-strand breaks (DSBs) that initiate meiotic recombination. Spo11 is the enzyme responsible for cleaving DNA and is found in a complex that also contains Ski8, Rec102, and Rec104. The Mre11/Rad50/Xrs2 complex is required for both DSB formation and DSB processing. In this article we investigate the functions of the remaining three proteins--Mer2, Mei4, and Rec114--with particular emphasis on Mer2. The Mer2 protein is present in vegetative cells, but it increases in abundance and becomes phosphorylated specifically during meiotic prophase. Mer2 localizes to distinct foci on meiotic chromosomes, with foci maximally abundant prior to the formation of synaptonemal complex. If DSB formation is blocked (e.g., by a spo11 mutation), dephosphorylation of Mer2 and its dissociation from chromosomes are delayed. We have also found that the Mei4 and Rec114 proteins localize to foci on chromosomes and these foci partially colocalize with each other and with Mer2. Furthermore, the three proteins co-immunoprecipitate. Mer2 does not show significant colocalization with Mre11 or Rec102 and Mer2 does not co-immunoprecipitate with Rec102. We propose that Mer2, Mei4, and Rec114 form a distinct complex required for DSB formation.

Chromosome Breakage↗
PubMed2005

Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34.

Ubiquitin chains linked via lysine 48 (K48) of ubiquitin mediate recognition of ubiquitinated proteins by the proteasome. However, the mechanisms underlying polymerization of this targeting signal on a substrate are unknown. Here we dissect this process using the cyclin-dependent kinase inhibitor Sic1 and its ubiquitination by the cullin-RING ubiquitin ligase SCF(Cdc4) and the ubiquitin-conjugating enzyme Cdc34. We show that Sic1 ubiquitination can be separated into two steps: attachment of the first ubiquitin, which is rate limiting, followed by rapid elongation of a K48-linked ubiquitin chain. Mutation of an acidic loop conserved among Cdc34 orthologs has no effect on attachment of the first ubiquitin onto Sic1 but compromises the processivity and linkage specificity of ubiquitin-chain synthesis. We propose that the acidic loop favorably positions K48 of a substrate-linked ubiquitin to attack SCF bound Cdc34 approximately ubiquitin thioester and thereby enables processive synthesis of K48-linked ubiquitin chains by SCF-Cdc34.

Anaphase-Promoting Complex-Cyclosome↗