Multisubunit Protein Complex Research Articles

Regulation of dynein-dynactin-driven vesicular transport.

Most of the long-range intracellular movements of vesicles, organelles and other cargoes are driven by microtubule (MT)-based molecular motors. Cytoplasmic dynein, a multisubunit protein complex, with the aid of dynactin, drives transport of a wide variety of cargoes towards the minus end of MTs. In this article, I review our current understanding of the mechanisms underlying spatiotemporal regulation of dynein-dynactin-driven vesicular transport with a special emphasis on the many steps of directional movement along MT tracks. These include the recruitment of dynein to MT plus ends, the activation and processivity of dynein, and cargo recognition and release by the motor complex at the target membrane. Furthermore, I summarize the most recent findings about the fine control mechanisms for intracellular transport via the interaction between the dynein-dynactin motor complex and its vesicular cargoes.

The Arabidopsis homolog of Scc4/MAU2 is essential for embryogenesis

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species cohesin is loaded on chromatin by the Scc2/Scc4 (NIBPL/MAU2) complex. Here we identify AtSCC4, the Arabidopsis homolog of Scc4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show suspensor over-proliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo . Our findings define Scc2/Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance and provide new insight into plant-specific role of this complex in controlling cell fate during embryogenesis.

Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs

The initial transfer of a complex glycan in protein N-glycosylation is catalyzed by oligosaccharyltransferase (OST), which is generally a multisubunit membrane protein complex in the endoplasmic reticulum but a single-subunit enzyme (ssOST) in some protists. To investigate the reaction mechanism of ssOST, we recombinantly expressed, purified and characterized the STT3A protein from Trypanosoma brucei (TbSTT3A). We analyzed the in vitro activity of TbSTT3A by synthesizing fluorescently labeled acceptor peptides as well as lipid-linked oligosaccharide (LLO) analogs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and C25) and with different double bond stereochemistry. We found that in addition to proline, charged residues at the +1 position of the sequon inhibited glycan transfer. An acidic residue at the −2 position significantly increased catalytic turnover but was not essential, in contrast to the bacterial OST. While all synthetic LLO analogs were processed by TbSTT3A, the length of the polyprenyl tail, but not the stereochemistry of the double bonds, determined their apparent affinity. We also synthesized phosphonate analogs of the LLOs, which were found to be competitive inhibitors of the reaction, although with lower apparent affinity to TbSTT3A than the active pyrophosphate analogs.

Biolayer interferometry of lipid nanodisc-reconstituted yeast vacuolar H+ -ATPase.

Vacuolar H+ -ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V-ATPase activity is regulated by reversible disassembly, resulting in cytosolic V1 -ATPase and membrane-integral V0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V1 V0 ND). We show that V1 V0 ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.

Misrouting of v-ATPase subunit V0a1 dysregulates lysosomal acidification in a neurodegenerative lysosomal storage disease model

Defective lysosomal acidification contributes to virtually all lysosomal storage disorders (LSDs) and to common neurodegenerative diseases like Alzheimer’s and Parkinson’s. Despite its fundamental importance, the mechanism(s) underlying this defect remains unclear. The v-ATPase, a multisubunit protein complex composed of cytosolic V1-sector and lysosomal membrane-anchored V0-sector, regulates lysosomal acidification. Mutations in the CLN1 gene, encoding PPT1, cause a devastating neurodegenerative LSD, INCL. Here we report that in Cln1−/− mice, which mimic INCL, reduced v-ATPase activity correlates with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Notably, treatment of Cln1−/− mice with a thioesterase (Ppt1)-mimetic, NtBuHA, ameliorated this defect. Our findings reveal an unanticipated role of Cln1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase function dysregulate lysosomal acidification in other LSDs and common neurodegenerative diseases.

Influence of Flexible “ω” on the Activity of E. coli RNA Polymerase: A Thermodynamic Analysis

The Escherichia coli RNA polymerase (RNAP) is a multisubunit protein complex containing the smallest subunit, ω. Despite the evolutionary conservation of ω and its role in assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of RNAP with the global chaperone protein GroEL. With an aim to get better insight into the structure and functional role of ω, we isolated a dominant negative mutant of ω (ω6), which is predominantly α-helical, in contrast to largely unstructured native ω, and then studied its assembly with reconstituted core1 (α2ββ′) by a biophysical approach. The mutant showed higher binding affinity compared to native ω. We observed that the interaction between core1 and ω6 is driven by highly negative enthalpy and a small but unfavorable negative entropy term. Extensive structural alteration in ω6 makes it more rigid, the plasticity of the interacting domain formed by ω6 and core1 is compromised, which may be responsible for the entropic cost. Such tight binding of the structured mutant (ω6) affects initiation of transcription. However, once preinitiated, the complex elongates the RNA chain efficiently. The initiation of transcription requires recognition of appropriate σ-factors by the core enzyme (core2: α2ββ′ω). We found that the altered core enzyme (α2ββ′ω6) with mutant ω showed a decrease in binding affinity to the σ-factors (σ70, σ32 and σ38) compared to that of the core enzyme containing native ω. In the absence of unstructured ω, the association of σ-factors to the core is less efficient, suggesting that the flexible native ω plays a direct role in σ-factor recruitment.

Porphyrin Induced Multimerization of Solution-State Proteins

Interactions between charged porphyrins and complimentary or similarly charged proteins provide important models systems for studies of electron transfer processes, artificial photosynthesis, and control of protein-protein interactions. Typically, the experimental results are analyzed and discussed assuming that the proteins exist in a monodisperse state. Here, we explored interaction of four solution-state proteins (horse heart cytochrome c, hen egg-white lysozyme, 3-heme c-type cytochrome PpcA from Geobacter sulfurreducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several cationic and anionic water-soluble derivatives of tetraphenylporphyrin. Combined small- and wide-angle X-ray scattering experiments revealed formation of multimers with a wide range of complex sizes. Thermodynamic interaction parameters and complex binding stoichiometries were established with isothermal calorimetry. Locations of porphyrin binding sites were determined with heteronuclear single quantum coherence (HSQC) and total correlation spectroscopy (TOCSY) NMRs for PpcA and cytochrome c, while covalent labeling shielding experiments followed by LC-MS analysis of tryptic digests were used to map ligand binding sites on cyt c4 and lysozyme surfaces. The obtained results demonstrate that multimerization of solution-state proteins by large water-soluble ligands appears to be a wide-spread phenomenon controlled by a delicate interplay of electrostatic and hydrophobic forces. Molecular level mapping of the binding sites allows us to build a theory explaining the size of the formed complexes and provides opportunities for targeted design and assembly of multi-subunit protein complexes.

Loss of α2δ-1 Calcium Channel Subunit Function Increases the Susceptibility for Diabetes.

Reduced pancreatic β-cell function or mass is the critical problem in developing diabetes. Insulin release from β-cells depends on Ca2+ influx through high voltage-gated Ca2+ channels (HVCCs). Ca2+ influx also regulates insulin synthesis and insulin granule priming and contributes to β-cell electrical activity. The HVCCs are multisubunit protein complexes composed of a pore-forming α1 and auxiliary β and α2δ subunits. α2δ is a key regulator of membrane incorporation and function of HVCCs. Here we show that genetic deletion of α2δ-1, the dominant α2δ subunit in pancreatic islets, results in glucose intolerance and diabetes without affecting insulin sensitivity. Lack of the α2δ-1 subunit reduces the Ca2+ currents through all HVCC isoforms expressed in β-cells equally in male and female mice. The reduced Ca2+ influx alters the kinetics and amplitude of the global Ca2+ response to glucose in pancreatic islets and significantly reduces insulin release in both sexes. The progression of diabetes in males is aggravated by a selective loss of β-cell mass, while a stronger basal insulin release alleviates the diabetes symptoms in most α2δ-1-/- female mice. Together, these findings demonstrate that the loss of the Ca2+ channel α2δ-1 subunit function increases the susceptibility for developing diabetes in a sex-dependent manner.

Mitochondrial malfunction in vanishing white matter disease: a disease of the cytosolic translation machinery.

Mitochondrial malfunction in vanishing white matter disease: a disease of the cytosolic translation machinery.

The Regulation and Function of Cohesin and Condensin in Mammalian Oocytes and Spermatocytes.

Germ cells, such as oocytes and spermatocytes, produce haploid gametes by a special type of cell division called meiosis. The reduction of chromosome number is achieved in meiosis I, in which homologous chromosomes (homologs) are paired and recombined with their counterparts and finally segregated from each other. How meiotic chromosomes behave in a different manner from mitotic chromosomes has been a fascinating problem for cellular and developmental biology. Cohesin and condensin are multi-subunit protein complexes that play central roles in sister chromatid cohesion and chromosome condensation (also segregation), respectively. Recent studies investigating the expression and function of cohesin and condensin in mammalian germ cells greatly advance our understanding of the molecular mechanism underlying the meiotic chromosomal events. Furthermore, accumulating evidence suggests that reduction of cohesin during prophase I arrest in mammalian oocytes is one of the major causes for age-related chromosome segregation error. This review focuses on the regulation and functions of cohesins and condensins during mammalian meiosis.

Photosynthetic water splitting by the Mn4Ca2+OX catalyst of photosystem II: its structure, robustness and mechanism.

The biological energy cycle of our planet is driven by photosynthesis whereby sunlight is absorbed by chlorophyll and other accessory pigments. The excitation energy is then efficiently transferred to a reaction centre where charge separation occurs in a few picoseconds. In the case of photosystem II (PSII), the energy of the charge transfer state is used to split water into oxygen and reducing equivalents. This is accomplished by the relatively low energy content of four photons of visible light. PSII is a large multi-subunit membrane protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Four high energy electrons, together with four protons (4H+), are used to reduce plastoquinone (PQ), the terminal electron acceptor of PSII, to plastoquinol (PQH2). PQH2 passes its reducing equivalents to an electron transfer chain which feeds into photosystem I (PSI) where they gain additional reducing potential from a second light reaction which is necessary to drive CO2 reduction. The catalytic centre of PSII consists of a cluster of four Mn ions and a Ca2+ linked by oxo bonds. In addition, there are seven amino acid ligands. In this Article, I discuss the structure of this metal cluster, its stability and the probability that an acid-base (nucleophilic-electrophilic) mechanism catalyses the water splitting reaction on the surface of the metal-cluster. Evidence for this mechanism is presented from studies on water splitting catalysts consisting of organo-complexes of ruthenium and manganese and also by comparison with the enzymology of carbon monoxide dehydrogenase (CODH). Finally the relevance of our understanding of PSII is discussed in terms of artificial photosynthesis with emphasis on inorganic water splitting catalysts as oxygen generating photoelectrodes.

The Arabidopsis homolog of Scc4/MAU2 is essential for embryogenesis.

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex that is crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species, cohesin is loaded on chromatin by the Scc2-Scc4 complex (also known as the NIBPL-MAU2 complex). Here, we identify the Arabidopsis homolog of Scc4, which we denote Arabidopsis thaliana (At)SCC4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway, and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show the suspensor overproliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo Our findings define the Scc2-Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance, and provide new insight into the plant-specific role of this complex in controlling cell fate during embryogenesis.

放射光X線結晶構造解析による光合成・光化学系IIの水分解・酸素発生機構の解明

Photosystem II （PSII） is a multi-subunit protein complex embedded in the thylakoid membrane and splits water molecules, producing electrons, protons and molecular oxygen in photosynthesis. We started to purify and crystallize PSII in 1990, which led to the first crystal structure analysis at a resolution of 3.7 Å in 2003 using SPring-8. The resolution of the structure was raised to 1.9 Å in 2011, which was considered a breakthrough in the filed of photosynthesis because, on the basis of this atomic resolution structure, it became possible to discuss the molecular mechanism of the water-splitting reaction. We are continuing our studies to obtain much more precise information on the oxygen-evolving complex （OEC） at the S1-state of the Kok cycle and to analyze the intermediate state structures using femtosecond X-ray pulses from XFEL of SACLA.

The Ccr4-Not Complex: Architecture and Structural Insights.

The Ccr4-Not complex is an essential multi-subunit protein complex that plays a fundamental role in eukaryotic mRNA metabolism and has a multitude of different roles that impact eukaryotic gene expression . It has a conserved core of three Not proteins, the Ccr4 protein, and two Ccr4 associated factors, Caf1 and Caf40. A fourth Not protein, Not4, is conserved, but is only a stable subunit of the complex in yeast. Certain subunits have been duplicated during evolution, with functional divergence, such as Not3 in yeast, and Ccr4 or Caf1 in human. However the complex includes only one homolog for each protein. In addition, species-specific subunits are part of the complex, such as Caf130 in yeast or Not10 and Not11 in human. Two conserved catalytic functions are associated with the complex, deadenylation and ubiquitination . The complex adopts an L-shaped structure, in which different modules are bound to a large Not1 scaffold protein. In this chapter we will summarize our current knowledge of the architecture of the complex and of the structure of its constituents.

Bioinformatics Analysis of PTM-Modified Protein Interaction Networks and Complexes.

Normal cellular functioning is maintained by macromolecular machines that control both core and specialized molecular tasks. These machines are in large part multi-subunit protein complexes that undergo regulation at multiple levels, from expression of requisite components to a vast array of post-translational modifications (PTMs). PTMs such as phosphorylation, ubiquitination, and acetylation currently number more than 200,000 in the human proteome and function within all molecular pathways. Here we provide a framework for systematically studying these PTMs in the context of global protein-protein interaction networks. This analytical framework allows insight into which functions specific PTMs tend to cluster in, and furthermore which complexes either single or multiple PTM signaling pathways converge on.

Effect of vacuolar ATPase subunit H (VmaH) on cellular pH, asexual cycle, stress tolerance and virulence in Beauveria bassiana

Vacuolar ATPase (V-ATPase) is a conserved multi-subunit protein complex that mediates intracellular acidification in fungi. Here we show functional diversity of V-ATPase subunit H (BbVmaH) in Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of BbvmaH resulted in elevated vacuolar pH, increased Ca2+ level in cytosol but not in vacuoles, accelerated culture acidification and reduced accumulation of extracellular ammonia. Aerial conidiation and submerged blastospore production were largely delayed and reduced in the deletion mutant, respectively, accompanied with a significant delay in conidial germination, alterations of conidia and blastospores in morphology, size and/or density, and severe growth defects in minimal media with different carbon and nitrogen sources. Despite null responses to osmotic, oxidative and cell wall perturbing stresses, the deletion mutant showed increased sensitivity to Ca2+, Zn2+ and Cu2+ during growth while its conidia were less tolerant to a wet-heat stress at 45°C and UV-B irradiation. Intracellular glycerol and mannitol contents also decreased significantly. Its virulence to Galleria mellonella larvae was significantly attenuated when conidia were topically applied for normal cuticle infection or injected into haemocoel for cuticle-bypassing infection. All phenotypic changes were restored by targeted gene complementation. Our results indicate that BbVmaH plays an important role in sustaining not only vacuolar acidification but also cytosolic calcium accumulation, ambient pH homeostasis, in vitro asexual cycle and virulence in B. bassiana.

The genome of Rhizobiales bacteria in predatory ants reveals urease gene functions but no genes for nitrogen fixation.

Gut-associated microbiota of ants include Rhizobiales bacteria with affiliation to the genus Bartonella. These bacteria may enable the ants to fix atmospheric nitrogen, but no genomes have been sequenced yet to test the hypothesis. Sequence reads from a member of the Rhizobiales were identified in the data collected in a genome project of the ant Harpegnathos saltator. We present an analysis of the closed 1.86 Mb genome of the ant-associated bacterium, for which we suggest the species name Candidatus Tokpelaia hoelldoblerii. A phylogenetic analysis reveals a relationship to Bartonella and Brucella, which infect mammals. Novel gene acquisitions include a gene for a putative extracellular protein of more than 6,000 amino acids secreted by the type I secretion system, which may be involved in attachment to the gut epithelium. No genes for nitrogen fixation could be identified, but genes for a multi-subunit urease protein complex are present in the genome. The urease genes are also present in Brucella, which has a fecal-oral transmission pathway, but not in Bartonella, which use blood-borne transmission pathways. We hypothesize that the gain and loss of the urease function is related to transmission strategies and lifestyle changes in the host-associated members of the Rhizobiales.

Structure of photosystem II and substrate binding at room temperature.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

Effect of Cross-linking on the Performance and Stability of Photocatalytic Photosystem I Films

Extracted from the chloroplasts of green plants, Photosystem I (PSI) is a multi-subunit protein complex that can be deposited as a thin film onto electrodes and incorporated into biohybrid photovoltaic devices to convert sunlight into usable energy. Previously, our group characterized persistent photocurrents over nine months using PSI multilayer films in an aqueous solution of redox mediators. However, the loss of periphery PSI proteins upon exposure to mediator solutions is known to affect the photocatalytic performance of the overall film. The cross-linker 2-iminothiolane (2IT) is capable of connecting protein molecules with covalent bonds, and is herein used to stabilize PSI multilayer films on gold substrates against significant desorption/degradation. Results showed that the thickness retainment of the cross-linked PSI films was superior to that of non-cross-linked films. PSI films modified by 2IT maintained production of photocurrent with long-term exposure to aqueous solutions in contrast to non-cross-linked control films.

WITHDRAWN: CSN1201, , a subunit of the COP9 signalosome, regulates the virulence in Cryptococcus neoformans infection

The COP9 signalosome (CSN) is a multisubunit protein complex, and it now has been found to participate in diverse cellular and developmental processes in various eukaryotic organisms. Cryptococcus neoformans (C. neoformans) is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immune compromised population. Here, we generated CSN deletion mutants to investigate the role in Cryptococcus infection. Compared to other CSN mutants, we identified a CSN1201 mutant exhibited severely attenuated virulence. Deletion of CSN1201 made cryptococcal cells more susceptible to nearly all in vitro stresses. Furthermore, deletion of CSN1201 obviously impaired survival of C. neoformans. At the same time, in vivo virulence assay of mouse infection models demonstrated that CSN1201 significantly enhanced the virulence of C. neoformans compared with the other CSN subunit strains, while ELISA analysis of C. neoformans infection in innate or adaptive immune response showed that deletion of CSN1201 significantly impaired cytokines and interferon expression. In vitro model of the blood-brain barrier (BBB) analysis indicated that deletion of CSN1201 reduced the invasion efficacy of Cryptococcusto cross BBB. Taken together, our findings reveal a novel mechanism of CSN1201, which plays a critical role for the virulence composite of C. neoformans, and also provides an additional yeast survival and propagation advantage in the host.