Core Subcomplexes Research Articles

Measurement of histone replacement dynamics with genetically encoded exchange timers in yeast.

Histone exchange between histones carrying position-specific marks and histones bearing general marks is important for gene regulation, but understanding of histone exchange remains incomplete. To overcome the poor time resolution of conventional pulse-chase histone labeling, we present a genetically encoded histone exchange timer sensitive to the duration that two tagged histone subunits co-reside at an individual genomic locus. We apply these sensors to map genome-wide patterns of histone exchange in yeast using single samples. Comparing H3 exchange in cycling and G1-arrested cells suggests that replication-independent H3 exchange occurs at several hundred nucleosomes (<1% of all nucleosomes) per minute, with a maximal rate at histone promoters. We observed substantial differences between the two nucleosome core subcomplexes: H2A-H2B subcomplexes undergo rapid transcription-dependent replacement within coding regions, whereas H3-H4 replacement occurs predominantly within promoter nucleosomes, in association with gene activation or repression. Our timers allow the in vivo study of histone exchange dynamics with minute time scale resolution.

Component Interaction of ESCRT Complexes Is Essential for Endocytosis-Dependent Growth, Reproduction, DON Production and Full Virulence in Fusarium graminearum.

Multivesicular bodies (MVBs) are critical intermediates in the trafficking of ubiquitinated endocytosed surface proteins to the lysosome/vacuole for destruction. Recognizing and packaging ubiquitin modified cargoes to the MVB pathway require ESCRT (Endosomal sorting complexes required for transport) machinery, which consists of four core subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. Our previous results showed that ESCRT-0 is essential for fungal development and pathogenicity in Fusarium graminearum. We then, in this study, systemically studied the protein-protein interactions within F. graminearum ESCRT-I, -II or -III complex, as well as between ESCRT-0 and ESCRT-I, ESCRT-I and ESCRT-II, and ESCRT-II and ESCRT-III complexes and found that loss of any ESCRT component resulted in abnormal function in endocytosis. In addition, ESCRT deletion mutants displayed severe defects in growth, deoxynivalenol (DON) production, virulence, sexual, and asexual reproduction. Importantly genetic complementation with corresponding ESCRT genes fully rescued all these defective phenotypes, indicating the essential role of ESCRT machinery in fungal development and plant infection in F. graminearum. Taken together, the protein-protein interactome and biological functions of the ESCRT machinery is first profoundly characterized in F. graminearum, providing a foundation for further exploration of ESCRT machinery in filamentous fungi.

TFIIH localization is highly dynamic during zygotic genome activation in Drosophila, and its depletion causes catastrophic mitosis.

In Drosophila, zygotic genome activation occurs in pre-blastoderm embryos during rapid mitotic divisions. How the transcription machinery is coordinated to achieve this goal in a very brief time span is still poorly understood. Transcription factor II H (TFIIH) is fundamental for transcription initiation by RNA polymerase II (RNAPII). Herein, we show the in vivo dynamics of TFIIH at the onset of transcription in Drosophila embryos. TFIIH shows an oscillatory behaviour between the nucleus and cytoplasm. TFIIH foci are observed from interphase to metaphase, and colocalize with those for RNAPII phosphorylated at serine 5 (RNAPIIS5P) at prophase, suggesting that transcription occurs during the first mitotic phases. Furthermore, embryos with defects in subunits of either the CAK or the core subcomplexes of TFIIH show catastrophic mitosis. Although, transcriptome analyses show altered expression of several maternal genes that participate in mitosis, the global level of RNAPIIS5P in TFIIH mutant embryos is similar to that in the wild type, therefore, a direct role for TFIIH in mitosis cannot be ruled out. These results provide important insights regarding the role of a basal transcription machinery component when the zygotic genome is activated.

Simultaneous Manipulation and Super-Resolution Fluorescence Imaging of Individual Kinetochores Coupled to Microtubule Tips.

Kinetochores are large multiprotein complexes that drive mitotic chromosome movements by mechanically coupling them to the growing and shortening tips of spindle microtubules. Kinetochores are also regulatory hubs, somehow sensing when they are erroneously attached and, in response, releasing their incorrect attachments and generating diffusible wait signals to delay anaphase until proper attachments can form. The remarkable ability of a kinetochore to sense and respond to its attachment status might stem from attachment- or tension-dependent changes in the structural arrangement of its core subcomplexes. However, direct tests of the relationship between attachment, tension, and core kinetochore structure have not previously been possible because of the difficulties of applying well-controlled forces and determining unambiguously the attachment status of individual kinetochores in vivo. The recent purification of native yeast kinetochores has enabled in vitro optical trapping-based assays of kinetochore tip-coupling and, in separate experiments, fluorescence imaging of single kinetochore particles. Here we introduce a dual instrument, combining optical trapping with multicolor total internal reflection fluorescence (TIRF) imaging, to allow kinetochore structure to be monitored directly with nanometer precision while mechanical tension is simultaneously applied. Our instrument incorporates differential interference contrast (DIC) imaging as well, to minimize the photo-bleaching of fluorescent tags during preparative bead and microtubule manipulations. A simple modification also allows the trapping laser to be easily converted into a real-time focus detection and correction system. Using this combined instrument, the distance between specific subcomplexes within a single kinetochore particle can be measured with 2-nm precision after 50s observation time, or with 11-nm precision at 1s temporal resolution. While our instrument was constructed specifically for studying kinetochores, it should also be useful for studying other filament-binding protein complexes, such as spindle poles, cortical microtubule attachments, focal adhesions, or other motor-cytoskeletal junctions.

Phycobilisome model with novel skeleton-like structures in a glaucocystophyte Cyanophora paradoxa

Phycobilisome (PBS) is a photosynthetic antenna supercomplex consisting of a central core subcomplex with several peripheral rods radiating from the core. Subunit structure of PBS was studied in a glaucocystophyte Cyanophora paradoxa strain NIES 547. Subunit composition of PBS was identified by N-terminal sequencing and genes for the subunits were determined by homology search of databases. They included rod linker proteins CpcK1 and CpcK2, rod-core linker proteins CpcG1 and CpcG2, and core linker proteins ApcC1 and ApcC2. Subfractionation by native polyacrylamide gel electrophoresis provided evidence for novel subcomplexes (ApcE/CpcK1/CpcG2/ApcA/ApcB/CpcD and ApcE/CpcK2/CpcG1/ApcA/ApcB), which connect rod and core subcomplexes. These skeleton-like structures may serve as a scaffold of the whole PBS assembly. Different roles of ApcC1 and ApcC2 were also suggested. Based on these findings, structural models for PBS were proposed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

The Mediator Complex in Plants: Structure, Phylogeny, and Expression Profiling of Representative Genes in a Dicot (Arabidopsis) and a Monocot (Rice) during Reproduction and Abiotic Stress

The Mediator (Med) complex relays regulatory information from DNA-bound transcription factors to the RNA polymerase II in eukaryotes. This macromolecular unit is composed of three core subcomplexes in addition to a separable kinase module. In this study, conservation of Meds has been investigated in 16 plant species representing seven diverse groups across the plant kingdom. Using Hidden Markov Model-based conserved motif searches, we have identified all the known yeast/metazoan Med components in one or more plant groups, including the Med26 subunits, which have not been reported so far for any plant species. We also detected orthologs for the Arabidopsis (Arabidopsis thaliana) Med32, -33, -34, -35, -36, and -37 in all the plant groups, and in silico analysis identified the Med32 and Med33 subunits as apparent orthologs of yeast/metazoan Med2/29 and Med5/24, respectively. Consequently, the plant Med complex appears to be composed of one or more members of 34 subunits, as opposed to 25 and 30 members in yeast and metazoans, respectively. Despite low similarity in primary Med sequences between the plants and their fungal/metazoan partners, secondary structure modeling of these proteins revealed a remarkable similarity between them, supporting the conservation of Med organization across kingdoms. Phylogenetic analysis between plant, human, and yeast revealed single clade relatedness for 29 Med genes families in plants, plant Meds being closer to human than to yeast counterparts. Expression profiling of rice (Oryza sativa) and Arabidopsis Med genes reveals that Meds not only act as a basal regulator of gene expression but may also have specific roles in plant development and under abiotic stress conditions.

Molecular architecture of the Nup84-Nup145C-Sec13 edge element in the nuclear pore complex lattice.

Nuclear pore complexes (NPCs) facilitate all nucleocytoplasmic transport. These massive protein assemblies are modular, with a stable structural scaffold supporting more dynamically attached components. The scaffold is made from multiple copies of the heptameric Y-complex and the heteromeric Nic96 complex. We demonstrated that members of these core subcomplexes specifically share an ACE1 fold with Sec31 of the COPII vesicle coat and proposed a lattice model for the NPC based on this commonality. Here we present the crystal structure of the heterotrimeric 134 kDa complex of Nup84-Nup145C-Sec13 of the Y-complex. The heterotypic ACE1 interaction of Nup84-Nup145C is analogous to the homotypic ACE1 interaction of Sec31 that forms COPII lattice edge elements and is inconsistent with the alternative “fence-like” NPC model. We construct a molecular model of the Y-complex and compare the architectural principles of COPII and NPC lattices.

Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex

The multisubunit Mediator (MED) complex bridges DNA-bound transcriptional regulators to the RNA polymerase II (PolII) initiation machinery. In yeast, the 25 MED subunits are distributed within three core subcomplexes and a separable kinase module composed of Med12, Med13 and the Cdk8-CycC pair thought to control the reversible interaction between MED and PolII by phosphorylating repeated heptapeptides within the Rpb1 carboxyl-terminal domain (CTD). Here, MED conservation has been investigated across the eukaryotic kingdom. Saccharomyces cerevisiae Med2, Med3/Pgd1 and Med5/Nut1 subunits are apparent homologs of metazoan Med29/Intersex, Med27/Crsp34 and Med24/Trap100, respectively, and these and other 30 identified human MED subunits have detectable counterparts in the amoeba Dictyostelium discoideum, indicating that none is specific to metazoans. Indeed, animal/fungal subunits are also conserved in plants, green and red algae, entamoebids, oomycetes, diatoms, apicomplexans, ciliates and the ‘deep-branching’ protists Trichomonas vaginalis and Giardia lamblia. Surprisingly, although lacking CTD heptads, T. vaginalis displays 44 MED subunit homologs, including several CycC, Med12 and Med13 paralogs. Such observations have allowed the identification of a conserved 17-subunit framework around which peripheral subunits may be assembled, and support a very ancient eukaryotic origin for a large, four-module MED. The implications of this comprehensive work for MED structure–function relationships are discussed.