RESUMEN
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Asunto(s)
Factor 4F Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Respuesta al Choque Térmico , Proteínas de Unión a Poli(A) , Biosíntesis de Proteínas , ARN Mensajero , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Respuesta al Choque Térmico/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Regulación Fúngica de la Expresión Génica , Unión Proteica , ARN de Hongos/metabolismo , ARN de Hongos/genéticaRESUMEN
Distinct morphologies of the mitochondrial network support divergent metabolic and regulatory processes that determine cell function and fate1-3. The mechanochemical GTPase optic atrophy 1 (OPA1) influences the architecture of cristae and catalyses the fusion of the mitochondrial inner membrane4,5. Despite its fundamental importance, the molecular mechanisms by which OPA1 modulates mitochondrial morphology are unclear. Here, using a combination of cellular and structural analyses, we illuminate the molecular mechanisms that are key to OPA1-dependent membrane remodelling and fusion. Human OPA1 embeds itself into cardiolipin-containing membranes through a lipid-binding paddle domain. A conserved loop within the paddle domain inserts deeply into the bilayer, further stabilizing the interactions with cardiolipin-enriched membranes. OPA1 dimerization through the paddle domain promotes the helical assembly of a flexible OPA1 lattice on the membrane, which drives mitochondrial fusion in cells. Moreover, the membrane-bending OPA1 oligomer undergoes conformational changes that pull the membrane-inserting loop out of the outer leaflet and contribute to the mechanics of membrane remodelling. Our findings provide a structural framework for understanding how human OPA1 shapes mitochondrial morphology and show us how human disease mutations compromise OPA1 functions.
Asunto(s)
GTP Fosfohidrolasas , Fusión de Membrana , Mitocondrias , Membranas Mitocondriales , Humanos , Biocatálisis , Cardiolipinas/química , Cardiolipinas/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Mitocondrias/química , Mitocondrias/metabolismo , Membranas Mitocondriales/química , Membranas Mitocondriales/enzimología , Membranas Mitocondriales/metabolismo , Mutación , Dominios Proteicos , Multimerización de Proteína , Dinámicas MitocondrialesRESUMEN
During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle1. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor2-4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline-arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair8-13 and chromatin organization14-17.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Anafase , Cromatina/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Microtúbulos/química , Microtúbulos/metabolismo , Membrana Nuclear/química , Huso Acromático/metabolismoRESUMEN
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs), which are formed from multiple copies of ~30 different nucleoporins (Nups) and inserted into the double nuclear membrane. Many of these Nups are organized into subcomplexes, of which the Y-shaped Nup84 complex is the major constituent of the nuclear and cytoplasmic rings. The Nup82-Nup159-Nsp1 complex is another module that, however, is only assembled into the cytoplasmic ring. By means of crosslinking mass spectrometry, biochemical reconstitution, and molecular modeling, we identified a short linear motif in the unstructured N-terminal region of Chaetomium thermophilum Nup145C, a subunit of the Y-complex, that is sufficient to recruit the Nup82 complex, but only in its assembled state. This finding points to a more general mechanism that short linear motifs in structural Nups can act as sensors to cooperatively connect pre-assembled NPC modules, thereby facilitating the formation and regulation of the higher-order NPC assembly.
Asunto(s)
Chaetomium/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Poro Nuclear/metabolismo , Secuencias de Aminoácidos , Chaetomium/química , Chaetomium/genética , Proteínas Fúngicas/genética , Modelos Moleculares , Poro Nuclear/química , Poro Nuclear/genética , Unión ProteicaRESUMEN
We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells. These custom-designed, photo-caged Q-rhodamines and fluoresceins are cell-permeable, bright and localize specifically to intracellular targets. We utilized established orthogonal protein labeling strategies to precisely attach the photoactivatable fluorophores to proteins with subsequent activation of fluorescence by irradiation with UV light. That way, diffusive cytosolic proteins, histone proteins as well as filigree mitochondrial networks and focal adhesion proteins were visualized inside living cells. We applied the new photoactivatable probes in inverse fluorescence recovery after photo-bleaching (iFRAP) experiments, gaining real-time access to protein dynamics from live biological settings with resolution in space and time. Finally, we used the caged Q-rhodamine for photo-activated localization microscopy (PALM) on both fixed and live mammalian cells, where the superior molecular brightness and photo-stability directly resulted in improved localization precisions for different protein targets.
RESUMEN
Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Alelos , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HeLa , Humanos , Proteínas de la Membrana/genética , Microscopía Fluorescente , Mitosis/genética , Modelos Biológicos , Proteínas Nucleares/genética , Fenotipo , Unión Proteica , Schizosaccharomyces/genética , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/genética , Eliminación de Secuencia , Imagen de Lapso de TiempoRESUMEN
Nuclear pore complexes (NPCs) are 110-megadalton assemblies that mediate nucleocytoplasmic transport. NPCs are built from multiple copies of ~30 different nucleoporins, and understanding how these nucleoporins assemble into the NPC scaffold imposes a formidable challenge. Recently, it has been shown how the Y complex, a prominent NPC module, forms the outer rings of the nuclear pore. However, the organization of the inner ring has remained unknown until now. We used molecular modeling combined with cross-linking mass spectrometry and cryo-electron tomography to obtain a composite structure of the inner ring. This architectural map explains the vast majority of the electron density of the scaffold. We conclude that despite obvious differences in morphology and composition, the higher-order structure of the inner and outer rings is unexpectedly similar.
Asunto(s)
Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Transporte Activo de Núcleo Celular , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Células HeLa , Humanos , Espectrometría de Masas , Modelos Moleculares , Matriz Nuclear/metabolismo , Matriz Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genéticaRESUMEN
Determining the structure of the nuclear pore complex (NPC) imposes an enormous challenge due to its size, intricate composition and membrane-embedded nature. In vertebrates, about 1000 protein building blocks assemble into a 110-MDa complex that fuses the inner and outer membranes of a cell's nucleus. Here, we review the recent progress in understanding the in situ architecture of the NPC with a specific focus on approaches using three-dimensional cryo electron microscopy. We discuss technological benefits and limitations and give an outlook toward obtaining a high-resolution structure of the NPC.
Asunto(s)
Poro Nuclear/fisiología , Animales , Microscopía por Crioelectrón/métodos , Humanos , Membranas/fisiología , Vertebrados/fisiologíaRESUMEN
Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/ultraestructura , Poro Nuclear/química , Poro Nuclear/ultraestructura , Sitios de Unión , Células HeLa , Humanos , Espectrometría de Masas , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/ultraestructura , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Conformación Proteica , Multimerización de Proteína , Estabilidad ProteicaRESUMEN
In eukaryotes, RNA Polymerase III (Pol III) is specifically responsible for transcribing genes encoding tRNAs and other short non-coding RNAs. The recruitment of Pol III to tRNA-encoding genes requires the transcription factors (TF) IIIB and IIIC. TFIIIC has been described as a conserved, multi-subunit protein complex composed of two subcomplexes, called τA and τB. How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood. Here we use chemical crosslinking mass spectrometry and determine the molecular architecture of TFIIIC. We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138. The identified τ131-τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation.
Asunto(s)
ARN Polimerasa III/metabolismo , Factores de Transcripción TFIII/metabolismo , Cristalografía por Rayos X , ARN Polimerasa III/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Masas en Tándem , Factores de Transcripción TFIII/químicaRESUMEN
Nuclear pore complexes (NPCs) are huge assemblies formed from â¼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82-Nup159-Nsp1-Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery.
Asunto(s)
Proteínas de Complejo Poro Nuclear/ultraestructura , Poro Nuclear/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Secuencia de Aminoácidos , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/ultraestructuraRESUMEN
Structural characterization of large multi-subunit protein complexes often requires integrating various experimental techniques. Cross-linking mass spectrometry (XL-MS) identifies proximal protein residues and thus is increasingly used to map protein interactions and determine the relative orientation of subunits within the structure of protein complexes. To fully adapt XL-MS as a structure characterization technique, we developed Xlink Analyzer, a software tool for visualization and analysis of XL-MS data in the context of the three-dimensional structures. Xlink Analyzer enables automatic visualization of cross-links, identifies cross-links violating spatial restraints, calculates violation statistics, maps chemically modified surfaces, and allows interactive manipulations that facilitate analysis of XL-MS data and aid designing new experiments. We demonstrate these features by mapping interaction sites within RNA polymerase I and the Rvb1/2 complex. Xlink Analyzer is implemented as a plugin to UCSF Chimera, a standard structural biology software tool, and thus enables seamless integration of XL-MS data with, e.g. fitting of X-ray structures to EM maps. Xlink Analyzer is available for download at http://www.beck.embl.de/XlinkAnalyzer.html.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Espectrometría de Masas/métodos , Proteínas/química , Programas Informáticos , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Reactivos de Enlaces Cruzados/química , ADN Helicasas/química , ADN Helicasas/metabolismo , Bases de Datos de Proteínas , Modelos Moleculares , Conformación Proteica , Proteínas/metabolismo , ARN Polimerasa I/química , ARN Polimerasa I/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.
Asunto(s)
Membrana Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Células HeLa , Humanos , Espectrometría de Masas , Modelos Moleculares , Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/ultraestructura , PolimerizacionRESUMEN
A key building block of the nuclear pore complex (NPC) is the Nup84 subcomplex that has been structurally analyzed predominantly in the yeast system. To expand this analysis and gain insight into the evolutionary conservation of its structure, we reconstituted an octameric Nup84 complex using the subunits from a thermophile, Chaetomium thermophilum (ct). This assembly carries Nup37 and Elys, which are characteristic subunits of the orthologous human Nup107-Nup160 complex but absent from the yeast Saccharomyces cerevisiae. We found that Elys binds cooperatively to the complex requiring both Nup37 and Nup120. Unexpectedly, the reconstituted ctNup84 complex formed a striking dimer structure with an unpredicted side-to-side arrangement of two molecules. Finally, crosslinking mass spectrometry allowed the mapping of key protein interfaces within the Y-shaped complex. Thus, the thermophilic Nup84 complex can serve as a structural model for higher eukaryotic Nup107-Nup160 assemblies to gain insight into its possible configuration within the NPC scaffold.