RESUMEN
The anaphase-promoting complex/cyclosome (APC/C) bound to CDC20 (APC/C(CDC20)) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) that inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which was proposed to require CDC20 autoubiquitylation. Here we characterize APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C's MCC binding site; it is required for APC/C-bound MCC (APC/C(MCC))-dependent CDC20 autoubiquitylation and degradation and for timely anaphase initiation but is dispensable for substrate ubiquitylation by APC/C(CDC20) and APC/C(CDH1). Our results support the model wherein MCC is continuously assembled and disassembled to enable rapid activation of APC/C(CDC20) and CDC20 autoubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators and report generation of recombinant human APC/C.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Modelos Biológicos , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Proteínas de Unión al Calcio/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Inmunoprecipitación , Puntos de Control de la Fase M del Ciclo Celular/genética , Proteínas Mad2 , Microscopía Electrónica , Microscopía Fluorescente , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Represoras/metabolismo , Imagen de Lapso de Tiempo , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
The multisubunit anaphase promoting complex (APC) is an essential cell-cycle regulator. Although CDC26 is known to have a role in APC assembly, its molecular function has remained unclear. Biophysical, structural and genetic studies presented here reveal that CDC26 stabilizes the structure of APC6, a core TPR protein required for APC integrity. Notably, CDC26-APC6 association involves an intermolecular TPR mimic composed of one helix from each protein.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Complejos de Ubiquitina-Proteína Ligasa/química , Ubiquitina-Proteína Ligasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dicroismo Circular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Covalent attachment of ubiquitin-like proteins (Ubls) is a predominant mechanism for regulating protein function in eukaryotes. Several structurally related Ubls, such as ubiquitin, SUMO, NEDD8, and ISG15, modify a vast number of proteins, altering their functions in a variety of ways. Ubl modifications can affect the target's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. Generally, these diverse Ubls are covalently attached via their C termini to their targets by parallel, but specific, cascades involving three classes of enzymes known as E1, E2, and E3. Structures are now available for many protein complexes in E1-E2-E3 cascades, revealing a series of modular building blocks and providing mechanistic insights into their functions.
Asunto(s)
Regulación de la Expresión Génica , Proteínas/química , Enzimas Activadoras de Ubiquitina/química , Ubiquitina/química , Animales , Biofisica/métodos , Humanos , Modelos Químicos , Conformación Molecular , Conformación Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Flock house virus (FHV) is the best-characterized member of the Nodaviridae, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.
Asunto(s)
Nodaviridae/genética , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/química , Animales , Células Cultivadas , Drosophila melanogaster , Transferencia Resonante de Energía de Fluorescencia , Inmunoprecipitación , Mediciones LuminiscentesRESUMEN
Positive-strand RNA viruses have proven to be valuable vectors for delivery and expression of antigens for direct vaccination of animals and vaccine production in plants. However, optimal use of these viruses as vectors for vaccine and other purposes is limited by incomplete understanding of their replication pathways and associated constraints on inserted foreign genes. Further insights into RNA virus vector design and optimization are emerging from recent advances on the function of viral RNA replication factors, the nature of the viral RNA replication complex as a membrane-bounded compartment sequestering replication components from competing processes and host defenses, and identification of surprisingly diverse host genes contributing to many virus replication steps.
Asunto(s)
Vectores Genéticos , Virus ARN/crecimiento & desarrollo , Virus ARN/genética , Replicación Viral/fisiología , Animales , Genómica , Humanos , Ensamble de VirusRESUMEN
Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.
Asunto(s)
Nucléolo Celular/metabolismo , Transcripción Genética , Secuencia de Bases , Línea Celular , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/ultraestructura , ADN/genética , Dactinomicina/farmacología , Metabolismo Energético , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Inmunoelectrónica , Proteínas Nucleares/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Factor de Empalme Asociado a PTB , ARN Nuclear/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína p14ARF Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions. In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells. Probability binning is also employed to provide a statistical confirmation of our observations. METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer. The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232). RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM. The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET. Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant. CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions. The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques. Its application to yeast presents a new method to a popular system widely used in proteomic studies.
Asunto(s)
Proteínas Bacterianas/química , Citometría de Flujo/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Proteínas Luminiscentes/química , Saccharomyces cerevisiae/metabolismo , Proteínas Bacterianas/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismoRESUMEN
Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.
Asunto(s)
Membranas Intracelulares/virología , Nodaviridae/genética , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/fisiología , Secuencia de Aminoácidos , Animales , Retículo Endoplásmico/virología , Ratones , Mitocondrias/virología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/fisiología , Levaduras/genética , Levaduras/crecimiento & desarrollo , Levaduras/ultraestructuraRESUMEN
PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.