RESUMO
Legionella pneumophila governs its interactions with host cells by secreting >300 different "effector" proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection.IMPORTANCELegionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in amoebae and macrophages by employing a "type IV" secretion system, which secretes more than 300 different "effector" proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.
Assuntos
Proteínas de Bactérias/genética , Evolução Molecular , Interações Hospedeiro-Patógeno , Legionella pneumophila/genética , Proteína ran de Ligação ao GTP/genética , Células A549 , Animais , Dictyostelium/microbiologia , Células HEK293 , Humanos , Legionella pneumophila/patogenicidade , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Disordered extensions at the termini and short internal insertions distinguish eukaryotic ribosomal proteins (r-proteins) from their anucleated archaeal counterparts. Here, we report an NMR structure of such a eukaryotic-specific segment (ESS) in the r-protein eS26 in complex with the escortin Tsr2. The structure reveals how ESS attracts Tsr2 specifically to importin:eS26 complexes entering the nucleus in order to trigger non-canonical RanGTP-independent disassembly. Tsr2 then sequesters the released eS26 and prevents rebinding to the importin, providing an alternative allosteric mechanism to terminate the process of nuclear import. Notably, a Diamond-Blackfan anemia-associated Tsr2 mutant protein is impaired in binding to ESS, unveiling a critical role for this interaction in human hematopoiesis. We propose that eS26-ESS and Tsr2 are components of a nuclear sorting system that co-evolved with the emergence of the nucleocytoplasmic barrier and transport carriers.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Carioferinas/metabolismo , Proteínas Ribossômicas/metabolismo , Transporte Ativo do Núcleo Celular , Sítio Alostérico , Núcleo Celular/metabolismo , Dicroísmo Circular , Citoplasma/metabolismo , Hematopoese , Humanos , Hibridização in Situ Fluorescente , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mutação , Proteínas Nucleares/metabolismo , Fenótipo , Ligação Proteica , Conformação Proteica , RNA/química , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.
Assuntos
Anemia de Diamond-Blackfan/patologia , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Anemia de Diamond-Blackfan/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Mutação de Sentido Incorreto , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome.
Assuntos
Adenilato Quinase/metabolismo , Substâncias Macromoleculares/metabolismo , Proteínas Nucleares/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Multimerização Proteica , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Accurate translation of the genetic code into functional polypeptides is key to cellular growth and proliferation. This essential process is carried out by the ribosome, a ribonucleoprotein complex of remarkable size and intricacy. Although the structure of the mature ribosome has provided insight into the mechanism of translation, our knowledge regarding the assembly, quality control, and intracellular targeting of this molecular machine is still emerging. Assembly of the eukaryotic ribosome begins in the nucleolus and requires more than 350 conserved assembly factors, which transiently associate with the preribosome at specific maturation stages. After accomplishing their tasks, early-acting assembly factors are released, preparing preribosomes for nuclear export. Export competent preribosomal subunits are transported through nuclear pore complexes into the cytoplasm, where they undergo final maturation steps, which are closely connected to quality control, before engaging in translation. In this chapter, we focus on the final events that commit correctly assembled ribosomal subunits for translation.
Assuntos
Núcleo Celular/metabolismo , Células Eucarióticas/metabolismo , Ribossomos/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Nucléolo Celular/metabolismo , Humanos , Proteínas Ribossômicas/metabolismoRESUMO
The transport receptor Crm1 mediates the export of diverse cargos containing leucine-rich nuclear export signals (NESs) through complex formation with RanGTP. To ensure efficient cargo release in the cytoplasm, NESs have evolved to display low affinity for Crm1. However, mechanisms that overcome low affinity to assemble Crm1-export complexes in the nucleus remain poorly understood. In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2. In vitro, Slx9 binds Rio2 and RanGTP, forming a complex. This complex directly loads Crm1, unveiling a non-canonical stepwise mechanism to assemble a Crm1-export complex. A mutation in Slx9 that impairs Crm1-export complex assembly inhibits 40S pre-ribosome export. Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export. This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.
Assuntos
Carioferinas/metabolismo , Complexos Multiproteicos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Epistasia Genética , Mutação/genética , Sinais de Exportação Nuclear , Ligação Proteica , Transporte Proteico , Ribossomos/metabolismo , Proteína ran de Ligação ao GTP/química , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1RESUMO
Within a single generation time a growing yeast cell imports â¼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Núcleo Celular/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Carioferinas/química , Carioferinas/genética , Carioferinas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estabilidade Proteica , Proteólise , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Ribossomos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , beta Carioferinas/química , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Construction of the eukaryotic ribosome begins in the nucleolus and requires >300 evolutionarily conserved nonribosomal trans-acting factors, which transiently associate with preribosomal subunits at distinct assembly stages. A subset of trans-acting and transport factors passage assembled preribosomal subunits in a functionally inactive state through the nuclear pore complexes (NPC) into the cytoplasm, where they undergo final maturation before initiating translation. Here, we summarize the repertoire of tools developed in the model organism budding yeast that are spearheading the functional analyses of trans-acting factors involved in the assembly and intracellular transport of preribosomal subunits. We elaborate on different GFP-tagged ribosomal protein reporters and a pre-rRNA reporter that reliably monitors the movement of preribosomal particles from the nucleolus to cytoplasm. We discuss the powerful yeast heterokaryon assay, which can be employed to uncover shuttling trans-acting factors that need to accompany preribosomal subunits to the cytoplasm to be released prior to initiating translation. Moreover, we present two biochemical approaches, namely sucrose gradient analyses and tandem affinity purification, that are rapidly facilitating the uncovering of regulatory processes that control the compositional dynamics of trans-acting factors on maturing preribosomal particles. Altogether, these approaches when combined with traditional analytical biochemistry, targeted proteomics and structural methodologies, will contribute to the dissection of the assembly and intracellular transport of preribosomal subunits, as well as other macromolecular assemblies that influence diverse biological pathways.
Assuntos
Poro Nuclear/metabolismo , Região Organizadora do Nucléolo/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico/genética , Proteínas de Fluorescência Verde/genética , Hibridização in Situ Fluorescente/métodos , Carioferinas/genética , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Poro Nuclear/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Receptores Citoplasmáticos e Nucleares/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Ultracentrifugação/métodos , Proteína Exportina 1RESUMO
Construction and intracellular targeting of eukaryotic pre-ribosomal particles involve a multitude of diverse transiently associating trans-acting assembly factors, energy-consuming enzymes, and transport factors. The ability to rapidly and reliably measure co-enrichment of multiple factors with maturing pre-ribosomal particles presents a major biochemical bottleneck towards revealing their function and the precise contribution of >50 energy-consuming steps that drive ribosome assembly. Here, we devised a workflow that combines genetic trapping, affinity-capture, and selected reaction monitoring mass spectrometry (SRM-MS), to overcome this deficiency. We exploited this approach to interrogate the dynamic proteome of pre-60S particles after nuclear export. We uncovered assembly factors that travel with pre-60S particles to the cytoplasm, where they are released before initiating translation. Notably, we identified a novel shuttling factor that facilitates nuclear export of pre-60S particles. Capturing and quantitating protein interaction networks of trapped intermediates of macromolecular complexes by our workflow is a reliable discovery tool to unveil dynamic processes that contribute to their in vivo assembly and transport.
Assuntos
Transporte Ativo do Núcleo Celular , Proteômica/métodos , Subunidades Ribossômicas Maiores de Eucariotos/química , Transporte Biológico , Espectrometria de Massas , Microscopia de Fluorescência , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Biogênese de Organelas , Mapas de Interação de Proteínas , Proteoma/análise , Proteoma/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The AAA-ATPase VCP (also known as p97) cooperates with distinct cofactors to process ubiquitylated proteins in different cellular pathways. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds to the plasma membrane protein caveolin-1 (CAV1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitylated CAV1 in SDS-resistant high-molecular-weight complexes on endosomes, which are en route to degradation in endolysosomes. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of CAV1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific caveolin-3 accumulates in sarcoplasmic pools and specifically delocalizes from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitylated cargo in the endocytic pathway and indicate that impaired trafficking of caveolin may contribute to pathogenesis in individuals with VCP mutations.