RESUMO
The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes.
Assuntos
Quinases Ciclina-Dependentes/ultraestrutura , Proteínas Quinases/ultraestrutura , RNA Polimerase II/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Transcrição Gênica , Sequência de Aminoácidos/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Cristalografia por Raios X , Quinases Ciclina-Dependentes/genética , Ciclinas/química , Ciclinas/ultraestrutura , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Fosforilação , Conformação Proteica , Proteínas Quinases/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Nuclear pore complexes (NPCs), which are composed of nucleoporins (Nups) and regulate transport between the nucleus and cytoplasm, significantly impact the replicative life span (RLS) of Saccharomyces cerevisiae We previously reported that deletion of the nonessential gene NUP100 increases RLS, although the molecular basis for this effect was unknown. In this study, we find that nuclear tRNA accumulation contributes to increased longevity in nup100Δ cells. Fluorescence in situ hybridization (FISH) experiments demonstrate that several specific tRNAs accumulate in the nuclei of nup100Δ mutants. Protein levels of the transcription factor Gcn4 are increased when NUP100 is deleted, and GCN4 is required for the elevated life spans of nup100Δ mutants, similar to other previously described tRNA export and ribosomal mutants. Northern blots indicate that tRNA splicing and aminoacylation are not significantly affected in nup100Δ cells, suggesting that Nup100 is largely required for nuclear export of mature, processed tRNAs. Distinct tRNAs accumulate in the nuclei of nup100Δ and msn5Δ mutants, while Los1-GFP nucleocytoplasmic shuttling is unaffected by Nup100. Thus, we conclude that Nup100 regulates tRNA export in a manner distinct from Los1 or Msn5. Together, these experiments reveal a novel Nup100 role in the tRNA life cycle that impacts the S. cerevisiae life span.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Núcleo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Complexo de Proteínas Formadoras de Poros Nucleares/genética , RNA Fúngico/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Northern Blotting , Divisão Celular , Meios de Cultura/química , Hibridização in Situ Fluorescente , Carioferinas/deficiência , Carioferinas/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , RNA Fúngico/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de TempoRESUMO
The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we find that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation.
Assuntos
Aquaporinas , Proteínas de Drosophila , Animais , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Cromatina , Origem de Replicação , Replicação do DNA , Drosophila/metabolismo , Aquaporinas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMO
The intricacy of nuclear pore complex (NPC) biogenesis imposes risks of failure that can cause defects in nuclear transport and nuclear envelope (NE) morphology; however, cellular mechanisms used to alleviate NPC assembly stress are not well defined. In the budding yeast Saccharomyces cerevisiae, we demonstrate that NVJ1- and MDM1-enriched NE-vacuole contacts increase when NPC assembly is compromised in several nup mutants, including nup116ΔGLFG cells. These interorganelle nucleus-vacuole junctions (NVJs) cooperate with lipid droplets to maintain viability and enhance NPC formation in assembly mutants. Additionally, NVJs function with ATG1 to remodel the NE and promote vacuole-dependent degradation of specific nucleoporins in nup116ΔGLFG cells. Importantly, NVJs significantly improve the physiology of NPC assembly mutants, despite having only negligible effects when NPC biogenesis is unperturbed. These results therefore define how NE-vacuole interorganelle contacts coordinate responses to mitigate deleterious cellular effects caused by disrupted NPC assembly.
Assuntos
Poro Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Deleção de Genes , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismoRESUMO
The eukaryotic nuclear permeability barrier and selective nucleocytoplasmic transport are maintained by nuclear pore complexes (NPCs), large structures composed of â¼ 30 proteins (nucleoporins [Nups]). NPC structure and function are disrupted in aged nondividing metazoan cells, although it is unclear whether these changes are a cause or consequence of aging. Using the replicative life span (RLS) of Saccharomyces cerevisiae as a model, we find that specific Nups and transport events regulate longevity independent of changes in NPC permeability. Mutants lacking the GLFG domain of Nup116 displayed decreased RLSs, whereas longevity was increased in nup100-null mutants. We show that Nup116 mediates nuclear import of the karyopherin Kap121, and each protein is required for mitochondrial function. Both Kap121-dependent transport and Nup116 levels decrease in replicatively aged yeast. Overexpression of GSP1, the small GTPase that powers karyopherin-mediated transport, rescued mitochondrial and RLS defects in nup116 mutants and increased longevity in wild-type cells. Together, these studies reveal that specific NPC nuclear transport events directly influence aging.