RESUMEN

Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.

Asunto(s)

Proteína BRCA2 , Daño del ADN , Reparación del ADN , Replicación del ADN , ADN de Cadena Simple , Recombinasa Rad51 , Xenopus laevis , Humanos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Animales , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Microscopía por Crioelectrón , ADN Polimerasa theta , Metilación de ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética

RESUMEN

During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled-coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full-length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80-Ndc80 interactions, prevent the formation of force-resistant kinetochore-microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore-microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop-mediated organization of adjacent Ndc80 complexes is crucial for stable end-on kinetochore-microtubule attachment and spindle assembly checkpoint satisfaction.

Asunto(s)

Cinetocoros , Microtúbulos , Segregación Cromosómica , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitosis , Unión Proteica , Animales

RESUMEN

POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors.

Asunto(s)

Replicación del ADN , Proteínas de Unión al ADN , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Proteínas de Unión al ADN/genética , Recombinación Homóloga/genética , ADN

RESUMEN

The oncogenic role of common fragile sites (CFS), focal and pervasive gaps in the cancer genome arising from replicative stress, remains controversial. Exploiting the TCGA dataset, we found that in most CFS the genes residing within the associated focal deletions are down-regulated, including proteins involved in tumour immune recognition. In a subset of CFS, however, the residing genes are surprisingly overexpressed. Within the most frequent CFS in this group, FRA4F, which is deleted in up to 18% of cancer cases and harbours the CCSER1 gene, we identified a region which includes an intronic, antisense pseudogene, TMSB4XP8. TMSB4XP8 focal ablation or transcriptional silencing elicits the overexpression of CCSER1, through a cis-acting mechanism. CCSER1 overexpression increases proliferation and triggers centrosome amplifications, multinuclearity, and aberrant mitoses. Accordingly, FRA4F is associated in patient samples to mitotic genes deregulation and genomic instability. As a result, cells overexpressing CCSER1 become sensitive to the treatment with aurora kinase inhibitors. Our findings point to a novel tumourigenic mechanism where focal deletions increase the expression of a new class of "dormant" oncogenes.

Asunto(s)

Proteínas de Ciclo Celular/genética , Sitios Frágiles del Cromosoma , Eliminación de Gen , Regulación hacia Arriba , Línea Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Mitosis , Seudogenes

RESUMEN

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

Asunto(s)

Proteína BRCA2/metabolismo , Replicación del ADN , ADN/biosíntesis , Recombinasa Rad51/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Proteína BRCA2/genética , Sitios de Unión , ADN/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Polimerasa I/metabolismo , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Femenino , Inestabilidad Genómica , Humanos , Proteína Homóloga de MRE11 , Masculino , Mutación , Unión Proteica , Recombinasa Rad51/genética , Origen de Réplica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Proteínas de Xenopus/genética , Xenopus laevis/genética

RESUMEN

Coordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double-strand break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA-damaging agents. Here, we examine the mechanistic aspects of HR-mediated fork protection, addressing the crosstalk between HR and replication proteins.

Asunto(s)

Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Recombinación Homóloga , Modelos Biológicos , Recombinasa Rad51/metabolismo , Ácido Anhídrido Hidrolasas , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Inestabilidad Cromosómica , Roturas del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína Homóloga de MRE11 , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Proteína de Replicación A/antagonistas & inhibidores , Proteína de Replicación A/química , Proteína de Replicación A/metabolismo

RESUMEN

The molecular mechanisms that underlie spleen development and congenital asplenia, a condition linked to increased risk of overwhelming infections, remain largely unknown. The transcription factor TLX1 controls cell fate specification and organ expansion during spleen development, and Tlx1 deletion causes asplenia in mice. Deregulation of TLX1 expression has recently been proposed in the pathogenesis of congenital asplenia in patients carrying mutations of the gene-encoding transcription factor SF-1. Herein, we have shown that TLX1-dependent regulation of retinoic acid (RA) metabolism is critical for spleen organogenesis. In a murine model, loss of Tlx1 during formation of the splenic anlage increased RA signaling by regulating several genes involved in RA metabolism. Uncontrolled RA activity resulted in premature differentiation of mesenchymal cells and reduced vasculogenesis of the splenic primordium. Pharmacological inhibition of RA signaling in Tlx1-deficient animals partially rescued the spleen defect. Finally, spleen growth was impaired in mice lacking either cytochrome P450 26B1 (Cyp26b1), which results in excess RA, or retinol dehydrogenase 10 (Rdh10), which results in RA deficiency. Together, these findings establish TLX1 as a critical regulator of RA metabolism and provide mechanistic insights into the molecular determinants of human congenital asplenia.

Asunto(s)

Proteínas de Homeodominio/fisiología , Transducción de Señal , Bazo/crecimiento & desarrollo , Tretinoina/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Femenino , Eliminación de Gen , Heterocigoto , Homocigoto , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación

RESUMEN

Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore-centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers.DOI: http://dx.doi.org/10.7554/eLife.02978.001.

Asunto(s)

Proteínas Cromosómicas no Histona/metabolismo , GTP Fosfohidrolasas/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Cristalografía por Rayos X , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Células HeLa , Humanos , Cinetocoros/química , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Pliegue de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína , ARN Interferente Pequeño/genética , Homología de Secuencia de Aminoácido

RESUMEN

Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.

Asunto(s)

Cinetocoros/química , Proteínas Asociadas a Microtúbulos/química , Secuencia de Aminoácidos , Centrómero/química , Segregación Cromosómica , Cristalografía por Rayos X , Escherichia coli/metabolismo , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Microscopía Electrónica , Microtúbulos/química , Mitosis , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B.

Asunto(s)

Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Tubercidina/análogos & derivados , Aurora Quinasa B , Aurora Quinasas , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteína A Centromérica , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Proteínas del Citoesqueleto , Células HeLa , Humanos , Nocodazol/farmacología , Proteínas Nucleares/metabolismo , Fosforilación , Transducción de Señal , Tubercidina/farmacología

RESUMEN

Kinetochores are proteinaceous scaffolds implicated in the formation of load-bearing attachments of chromosomes to microtubules during mitosis. Kinetochores contain distinct chromatin- and microtubule-binding interfaces, generally defined as the inner and outer kinetochore, respectively (reviewed in). The constitutive centromere-associated network (CCAN) and the Knl1-Mis12-Ndc80 complexes (KMN) network are the main multisubunit protein assemblies in the inner and outer kinetochore, respectively. The point of contact between the CCAN and the KMN network is unknown. Cenp-C is a conserved CCAN component whose central and C-terminal regions have been implicated in chromatin binding and dimerization. Here, we show that a conserved motif in the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex. Expression in HeLa cells of the isolated N-terminal motif of Cenp-C prevents outer kinetochore assembly, causing chromosome missegregation. The KMN network is also responsible for kinetochore recruitment of the components of the spindle assembly checkpoint, and we observe checkpoint impairment in cells expressing the Cenp-C N-terminal segment. Our studies unveil a crucial and likely universal link between the inner and outer kinetochore.

Asunto(s)

Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Immunoblotting , Microscopía Electrónica , Microscopía Fluorescente , Plásmidos/genética

RESUMEN

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas Mad2 , Modelos Biológicos , Proteínas Nucleares/aislamiento & purificación , Unión Proteica , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación

RESUMEN

BACKGROUND: Mad1 and Mad2 are constituents of the spindle-assembly checkpoint, a device coupling the loss of sister-chromatid cohesion at anaphase to the completion of microtubule attachment of the sister chromatids at metaphase. Fluorescence recovery after photobleaching (FRAP) revealed that the interaction of cytosolic Mad2 with kinetochores is highly dynamic, suggesting a mechanism of catalytic activation of Mad2 at kinetochores followed by its release in a complex with Cdc20. The recruitment of cytosolic Mad2 to kinetochores has been attributed to a stable receptor composed of a distinct pool of Mad2 tightly bound to Mad1. Whether specifically this interaction accounts for the kinetochore dynamics of Mad2 is currently unknown. RESULTS: To gain a precise molecular understanding of the interaction of Mad2 with kinetochores, we reconstituted the putative Mad2 kinetochore receptor and developed a kinetochore recruitment assay with purified components. When analyzed by FRAP in vitro, this system faithfully reproduced the previously described in vivo dynamics of Mad2, providing an unequivocal molecular account of the interaction of Mad2 with kinetochores. Using the same approach, we dissected the mechanism of action of p31(comet), a spindle-assembly checkpoint inhibitor. CONCLUSIONS: In vitro FRAP is a widely applicable approach to dissecting the molecular bases of the interaction of a macromolecule with an insoluble cellular scaffold. The combination of in vitro fluorescence recovery after photobleaching with additional fluorescence-based assays in vitro can be used to unveil mechanism, stoichiometry, and kinetic parameters of a macromolecular interaction, all of which are important for modeling protein interaction networks.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Cinetocoros/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Unión al Calcio/química , Proteínas Cdc20 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiología , Proteínas Mad2 , Proteínas Nucleares/metabolismo , Proteínas Represoras/química , Huso Acromático/metabolismo

RESUMEN

BACKGROUND: The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands. RESULTS: We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC. CONCLUSIONS: We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Genes cdc/fisiología , Modelos Biológicos , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Huso Acromático/fisiología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Citosol/metabolismo , Escherichia coli , Citometría de Flujo , Células HeLa , Humanos , Inmunoprecipitación , Isomerismo , Cinetocoros/metabolismo , Proteínas Mad2 , Proteínas Nucleares , Plásmidos/genética , Interferencia de ARN

RESUMEN

We here report on the identification and detailed biochemical characterization of two novel GTPase-activating proteins, Gyp5p and Gyp8p, whose efficient substrate is Ypt1p, a Ypt/Rab-GTPase essential for endoplasmic reticulum-to-Golgi trafficking in yeast. Gyp5p accelerated the intrinsic GTPase activity of Ypt1p 4.2 x 10(4)-fold and, surprisingly, the 40-fold reduced GTP hydrolysis rate of Ypt1(Q67L)p 1.5 x 10(4)-fold. At steady state, the two newly discovered GTPase-activating proteins (GAPs) as well as the previously described Gyp1p, which also uses Ypt1p as the preferred substrate, display different subcellular localization. To add to an understanding of the significance of Ypt1p-bound GTP hydrolysis in vivo, yeast strains expressing the GTPase-deficient Ypt1(Q67L)p and having different Ypt1-GAP genes deleted were created. Depending on the genetic background, different mutants exhibited growth defects at low temperature and, already at permissive temperature, various morphological alterations resembling autophagy. Transport of proteins was not significantly impaired. Growth defects of Ypt1(Q67L)-expressing cells could be suppressed on high expression of all three Ypt1-GAPs. We propose that permanently active Ypt1p leads to increased vesicle fusion, which might induce previously unnoticed autophagic degradation of exaggerated membrane-enclosed structures. The data indicate that hydrolysis of Ypt1p-bound GTP is a prerequisite for a balanced vesicle flow between endoplasmic reticulum and Golgi compartments.

Asunto(s)

Retículo Endoplásmico/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Aparato de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Autofagia , Secuencia de Bases , Cartilla de ADN , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Hidrólisis , Microscopía Electrónica , Datos de Secuencia Molecular , Transporte de Proteínas , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Homología de Secuencia de Aminoácido

RESUMEN

The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2-Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1-Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled-coil to form two connected sub-complexes with Mad2. The Mad2 C-terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular 'safety belts'. We show that Mad1 is a competitive inhibitor of the Mad2-Cdc20 complex, and propose that the Mad1-Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1-Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1-Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1-Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C-terminal tail is required for ligand release from Mad2, and that the 'safety belt' mechanism may prolong the lifetime of Mad2-ligand complexes.

Asunto(s)

Proteínas de Unión al Calcio/química , Proteínas Portadoras , Proteínas Fúngicas/química , Fosfoproteínas/química , Proteínas Represoras/química , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido