RESUMEN
The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/química , Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Citidina Trifosfato/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células HeLa , Humanos , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
Signal transduction by small ubiquitin-like modifier (SUMO) regulates a myriad of nuclear processes. Here we report on the role of SUMO in mitosis in human cell lines. Knocking down the SUMO conjugation machinery results in a delay in mitosis and defects in mitotic chromosome separation. Searching for relevant SUMOylated proteins in mitosis, we identify the anaphase-promoting complex/cyclosome (APC/C), a master regulator of metaphase to anaphase transition. The APC4 subunit is the major SUMO target in the complex, containing SUMO acceptor lysines at positions 772 and 798. SUMOylation is crucial for accurate progression of cells through mitosis and increases APC/C ubiquitylation activity toward a subset of its targets, including the newly identified target KIF18B. Combined, our findings demonstrate the importance of SUMO signal transduction for genome integrity during mitotic progression and reveal how SUMO and ubiquitin cooperate to drive mitosis.
Asunto(s)
Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Mitosis/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación/genética , Ubiquitinas/metabolismo , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/genética , Línea Celular Tumoral , Células HCT116 , Células HeLa , Humanos , Cinesinas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/genética , Ubiquitinación/genética , Ubiquitinas/genéticaRESUMEN
The Anaphase Promoting Complex/Cyclosome (APC/C) is a ubiquitin E3 ligase that functions as the gatekeeper to mitotic exit. APC/C activity is controlled by an interplay of multiple pathways during mitosis, including the spindle assembly checkpoint (SAC), that are not yet fully understood. Here, we show that sumoylation of the APC4 subunit of the APC/C peaks during mitosis and is critical for timely APC/C activation and anaphase onset. We have also identified a functionally important SUMO interacting motif in the cullin-homology domain of APC2 located near the APC4 sumoylation sites and APC/C catalytic core. Our findings provide evidence of an important regulatory role for SUMO modification and binding in affecting APC/C activation and mitotic exit.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas del Citoesqueleto/genética , Mitosis/genética , Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/química , Dominio Catalítico/genética , Proteínas del Citoesqueleto/química , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo Celular/genética , Plásmidos/genética , Unión Proteica , Conformación Proteica , Huso Acromático/química , Huso Acromático/genética , Sumoilación/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5(N)). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5(N) has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5(N) shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes.
Asunto(s)
Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Subunidad Apc5 del Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Subunidades de Proteína/metabolismo , Animales , Ciclo Celular/fisiología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Estructura Terciaria de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces , Xenopus laevisRESUMEN
UNLABELLED: Human cytomegalovirus (HCMV) deregulates the cell cycle by several means, including inactivation of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Viral proteins UL97 and UL21a, respectively, affect the APC/C by phosphorylation of APC/C coactivator Cdh1 and by inducing the degradation of subunits APC4 and APC5, which along with APC1 form the APC/C platform subcomplex. The aim of this study was to further characterize the mechanism of APC/C inactivation and define the relative contributions of UL21a and UL97 to APC/C substrate accumulation and to viral growth. We show that in uninfected cells, UL21a but not UL97 can disrupt APC/C function, leading to the accumulation of substrates. We find that UL21a is necessary and sufficient to induce the degradation of APC1, in addition to the previously reported APC4 and APC5. We also demonstrate that there is a previously unreported cellular mechanism for a specific decrease in the levels of all three platform subunits, APC1, APC4, and APC5, upon the depletion of any one of these subunits or of subunit APC8. Finally, we show that at a low multiplicity of infection, either UL97 or UL21a can partially complement a growth-defective mutant virus lacking both UL21a and UL97, with significantly greater benefit afforded by the expression of both proteins. This double mutant also can be partially rescued by inactivation of the APC/C using small interfering RNAs against specific subunits. These results further our understanding of HCMV's interaction with the cell cycle machinery and reveal a new cellular pattern of APC/C subunit downmodulation. IMPORTANCE: HCMV lytic infection subverts the host cell cycle machinery in multiple ways. A major effect is inactivation of the APC/C, which plays a central role in the control of cell cycle progression. This study provides further insight into the mechanism of inactivation. We discovered that the APC1 subunit, which along with APC4 and APC5 form the platform subcomplex of the APC/C, is an additional target of the degradation induced by HCMV protein UL21a. This study also shows for the first time that there is a unique cellular process in uninfected cells whereby depletion of APC1, APC4, APC5, or APC8 recapitulates the pattern of HCMV-mediated APC/C subunit degradation.
Asunto(s)
Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Subunidad Apc5 del Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Citomegalovirus/fisiología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Proteínas Virales/metabolismo , Replicación Viral , Células Cultivadas , Interacciones Huésped-Patógeno , HumanosRESUMEN
Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.
Asunto(s)
Terapia Genética , Microcefalia/genética , Microcefalia/terapia , Células-Madre Neurales/fisiología , Proteínas Nucleares/deficiencia , Adenoviridae/genética , Animales , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Apoptosis/genética , Encéfalo/patología , Proteínas Portadoras/genética , Moléculas de Adhesión Celular/metabolismo , Ciclo Celular , Proliferación Celular , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Embrión de Mamíferos , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Microcefalia/patología , Nestina/genética , Nestina/metabolismo , Neurogénesis , Proteínas Nucleares/genética , Sinapsinas/genética , Sinapsinas/metabolismoRESUMEN
Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.
Asunto(s)
Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Poliubiquitina/biosíntesis , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/fisiología , Secuencia de Aminoácidos , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Puntos de Control del Ciclo Celular , Células HeLa , Humanos , Datos de Secuencia Molecular , Poliubiquitina/genética , Estructura Terciaria de ProteínaRESUMEN
MicroRNA-452 (miRNA-452) was overexpressed in docetaxel-resistant human breast cancer MCF-7 cells (MCF-7/DOC). However, its role in modulating the sensitivity of breast cancer cells to docetaxel (DOC) remains unclear. The aim of this study is to investigate the role of miRNA-452 in the sensitivity of breast cancer cells to DOC.Real-time quantitative PCR (RT-qPCR) were used to identify the differential expression of miRNA-452 between MCF-7/DOC and MCF-7 cells. MiRNA-452 mimic was transfected into MCF-7 cells and miRNA-452 inhibitor was transfected into MCF-7/DOC cells. The role of miRNA-452 in these transfected cells was evaluated using RT-qPCR, MTT assay, and flow cytometry assay. The relationship of miRNA-452 and its predictive target gene "anaphase-promoting complex 4" (APC4) was analyzed by RT-qPCR and Western blot.MiRNA-452 showed significantly higher expression (78.9-folds) in MCF-7/DOC cells compared to parental MCF-7 cells. The expression of miRNA-452 in the mimic transfected MCF-7 cells was upregulated 212.2-folds (P < 0.05) compared to its negative control (NC), and the half maximal inhibitory concentration (IC50) value of DOC (1.98 ± 0.15 µM) was significantly higher than that in its NC (0.85 ± 0.08 µM, P < 0.05) or blank control (1.01 ± 0.19 µM, P < 0.05). Furthermore, its apoptotic rate (6.3 ± 1.3 %) was distinctly decreased compared with that in its NC (23.8 ± 6.6 %, P < 0.05) or blank control (18.6 ± 4.7 %, P < 0.05). In contrast, the expression of miRNA-452 in the inhibitor-transfected MCF-7/DOC cells was downregulated 0.58-fold (P < 0.05) compared to its NC, the IC50 value of DOC (44.5 ± 3.2 µM) was significantly lower than that in its NC (107.3 ± 6.63 µM, P < 0.05) or blank control (102.22 ± 11.34 µM, P < 0.05), and the apoptotic rate (45.5 ± 10.8 %) was distinctly increased compared with its NC (9.9 ± 2.2 %, P < 0.05) and blank control (9.4 ± 2.5 %, P < 0.05). Further, there was an inverse association between miRNA-452 and APC4 expression in breast cancer cells in vitro.Dysregulation of miRNA-452 involved in the DOC resistance formation of breast cancer cells may be, in part, via targeting APC4.
Asunto(s)
Neoplasias de la Mama/genética , MicroARNs/genética , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/genética , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Docetaxel , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , MicroARNs/biosíntesis , Taxoides/administración & dosificaciónRESUMEN
The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109-110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Citomegalovirus/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas Virales/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc5 del Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Citomegalovirus/genética , Células HEK293 , Células HeLa , Humanos , Fosforilación , Unión Proteica , Eliminación de Secuencia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Virales/genéticaRESUMEN
Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G(1)/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.
Asunto(s)
Cadherinas/metabolismo , Infecciones por Citomegalovirus/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Antígenos CD , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc5 del Ciclosoma-Complejo Promotor de la Anafase , Secuencia de Bases , Sitios de Unión/genética , Cadherinas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/patogenicidad , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Cartilla de ADN/genética , Geminina , Eliminación de Gen , Genes Inmediatos-Precoces , Genes Virales , Humanos , Datos de Secuencia Molecular , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Inhibidores de Proteasoma , Estabilidad Proteica , Subunidades de Proteína , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Controls of stem cell maintenance and early differentiation are known in several systems. However, the progression from stem cell self-renewal to overt signs of early differentiation is a poorly understood but important problem in stem cell biology. The Caenorhabditis elegans germ line provides a genetically defined model for studying that progression. In this system, a single-celled mesenchymal niche, the distal tip cell (DTC), employs GLP-1/Notch signaling and an RNA regulatory network to balance self-renewal and early differentiation within the "mitotic region," which continuously self-renews while generating new gametes. Here, we investigate germ cells in the mitotic region for their capacity to differentiate and their state of maturation. Two distinct pools emerge. The "distal pool" is maintained by the DTC in an essentially uniform and immature or "stem cell-like" state; the "proximal pool," by contrast, contains cells that are maturing toward early differentiation and are likely transit-amplifying cells. A rough estimate of pool sizes is 30-70 germ cells in the distal immature pool and approximately 150 in the proximal transit-amplifying pool. We present a simple model for how the network underlying the switch between self-renewal and early differentiation may be acting in these two pools. According to our model, the self-renewal mode of the network maintains the distal pool in an immature state, whereas the transition between self-renewal and early differentiation modes of the network underlies the graded maturation of germ cells in the proximal pool. We discuss implications of this model for controls of stem cells more broadly.
Asunto(s)
Caenorhabditis elegans/citología , Células Germinativas/citología , Animales , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Femenino , Células Germinativas/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Mitosis , Modelos Biológicos , Mutación , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal , TemperaturaRESUMEN
Schizosaccharomyces pombe Dim1p is required for maintaining the steady-state level of the anaphase-promoting complex or cyclosome (APC/C) component Lid1p and thus for maintaining the steady-state level and activity of the APC/C. To gain further insight into Dim1p function, we have investigated the mechanism whereby Dim1p influences Lid1p levels. We show that S. pombe cells lacking Dim1p or Saccharomyces cerevisiae cells lacking its ortholog, Dib1p, are defective in generalized pre-mRNA splicing in vivo, a result consistent with the identification of Dim1p as a component of the purified yeast U4/U6.U5 tri-snRNP complex. Moreover, we find that Dim1p is part of a complex with the splicing factor Prp1p. However, although Dim1p is required for efficient splicing of lid1(+) pre-mRNA, circumventing the necessity for this particular function of Dim1p is insufficient for restoring normal Lid1p levels. Finally, we provide evidence that Dim1p also participates in the nuclear export of lid1(+) mRNA and that it is likely the combined loss of both of these two Dim1p functions which compromises Lid1p levels in the absence of proper Dim1p function. These data indicate that a mechanism acting at the level of mRNA impacts the functioning of the APC/C, a critical complex in controlling mitotic progression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase , Proteínas de Ciclo Celular/genética , Humanos , Sustancias Macromoleculares , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Técnicas del Sistema de Dos Híbridos , Complejos de Ubiquitina-Proteína Ligasa/químicaRESUMEN
BACKGROUND: Chromosome segregation and mitotic exit depend on activation of the anaphase-promoting complex (APC) by the substrate adaptor proteins CDC20 and CDH1. The APC is a ubiquitin ligase composed of at least 11 subunits. The interaction of APC2 and APC11 with E2 enzymes is sufficient for ubiquitination reactions, but the functions of most other subunits are unknown. RESULTS: We have biochemically characterized subcomplexes of the human APC. One subcomplex, containing APC2/11, APC1, APC4, and APC5, can assemble multiubiquitin chains but is unable to bind CDH1 and to ubiquitinate substrates. The other subcomplex contains all known APC subunits except APC2/11. This subcomplex can recruit CDH1 but fails to support any ubiquitination reaction. In vitro, the C termini of CDC20 and CDH1 bind to the closely related TPR subunits APC3 and APC7. Homology modeling predicts that these proteins are similar in structure to the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. APC activation by CDH1 depends on a conserved C-terminal motif that is also found in CDC20 and APC10. CONCLUSIONS: APC1, APC4, and APC5 may connect APC2/11 with TPR subunits. TPR domains in APC3 and APC7 recruit CDH1 to the APC and may thereby bring substrates into close proximity of APC2/11 and E2 enzymes. In analogy to PEX5, the different TPR subunits of the APC might function as receptors that interact with the C termini of regulatory proteins such as CDH1, CDC20, and APC10.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Enzimas Activadoras de Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc5 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc7 del Ciclosoma-Complejo Promotor de la Anafase , Western Blotting , Proteínas Cdc20 , Electroforesis en Gel de Poliacrilamida , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia , Tinción con Nitrato de PlataRESUMEN
Here we show that emb-30 is required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. Germline-specific emb-30 mutant alleles block the meiotic divisions. Mutant oocytes, fertilized by wild-type sperm, set up a meiotic spindle but do not progress to anaphase I. As a result, polar bodies are not produced, pronuclei fail to form, and cytokinesis does not occur. Severe-reduction-of-function emb-30 alleles (class I alleles) result in zygotic sterility and lead to germline and somatic defects that are consistent with an essential role in promoting the metaphase-to-anaphase transition during mitosis. Analysis of the vulval cell lineages in these emb-30(class I) mutant animals suggests that mitosis is lengthened and eventually arrested when maternally contributed emb-30 becomes limiting. By further reducing maternal emb-30 function contributed to class I mutant animals, we show that emb-30 is required for the metaphase-to-anaphase transition in many, if not all, cells. Metaphase arrest in emb-30 mutants is not due to activation of the spindle assembly checkpoint but rather reflects an essential emb-30 requirement for M-phase progression. A reduction in emb-30 activity can suppress the lethality and sterility caused by a null mutation in mdf-1, a component of the spindle assembly checkpoint machinery. This result suggests that delaying anaphase onset can bypass the spindle checkpoint requirement for normal development. Positional cloning established that emb-30 encodes the likely C. elegans orthologue of APC4/Lid1, a component of the anaphase-promoting complex/cyclosome, required for the metaphase-to-anaphase transition. Thus, the anaphase-promoting complex/cyclosome is likely to be required for all metaphase-to-anaphase transitions in a multicellular organism.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/citología , Proteínas de Ciclo Celular/metabolismo , Ligasas/genética , Proteínas de Saccharomyces cerevisiae , Complejos de Ubiquitina-Proteína Ligasa , Alelos , Secuencia de Aminoácidos , Anafase/fisiología , Ciclosoma-Complejo Promotor de la Anafase , Animales , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase , Caenorhabditis elegans/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Metafase/fisiología , Datos de Secuencia Molecular , Mutación , Oocitos/fisiología , Alineación de Secuencia , Ubiquitina-Proteína LigasasRESUMEN
The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8). The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned. APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27. Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G1 phase cyclins and cyclin-dependent kinase inhibitors. The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p. APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation.