RESUMO
The yeast [PSI+] prion is an epigenetic modifier of translation termination fidelity that causes nonsense suppression. The prion [PSI+] forms when the translation termination factor Sup35p adopts a self-propagating conformation. The presence of the [PSI+] prion modulates survivability in a variety of growth conditions. Nonsense suppression is essential for many [PSI+]-mediated phenotypes, but many do not appear to be due to read-through of a single stop codon, but instead are multigenic traits. We hypothesized that other global mechanisms act in concert with [PSI+] to influence [PSI+]-mediated phenotypes. We have identified one such global regulator, the Paf1 complex (Paf1C). Paf1C is conserved in eukaryotes and has been implicated in several aspects of transcriptional and posttranscriptional regulation. Mutations in Ctr9p and other Paf1C components reduced [PSI+]-mediated nonsense suppression. The CTR9 deletion also alters nonsense suppression afforded by other genetic mutations but not always to the same extent as the effects on [PSI+]-mediated read-through. Our data suggest that the Paf1 complex influences mRNA translatability but not solely through changes in transcript stability or abundance. Finally, we demonstrate that the CTR9 deletion alters several [PSI+]-dependent phenotypes. This provides one example of how [PSI+] and genetic modifiers can interact to uncover and regulate phenotypic variability.
Assuntos
Complexos Multiproteicos/metabolismo , Mutação/genética , Proteínas Nucleares/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alelos , Proteínas de Ciclo Celular/metabolismo , Códon sem Sentido/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Recessivos , Teste de Complementação Genética , Fatores de Terminação de Peptídeos , Fenótipo , Príons/química , Biossíntese de Proteínas , Estrutura Quaternária de Proteína , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Supressão Genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Elongação da Transcrição/deficiência , Fatores de Elongação da Transcrição/metabolismoRESUMO
Spindle pole body (SPB; the microtubule organizing center in yeast) duplication is essential to form a bipolar spindle. The duplicated SPBs must then separate and migrate to opposite sides of the nucleus. We identified a novel functional relationship in SPB separation between the microtubule stabilizing protein Bik1p/CLIP-170 and the SPB half-bridge protein Sfi1p. A genetic interaction between BIK1 and SFI1 was discovered in a synthetic lethal screen using a strain deficient in the prion protein gene RNQ1. RNQ1 deletion reduced expression from the divergently transcribed BIK1, allowing us to identify genetic interactors with bik1. The sfi1-1 bik1 synthetic lethality was suppressed by over-expression of CIK1, KAR1, and PPH21. Genetic analysis indicated that the sfi1-1 bik1 synthetic lethality was unlikely related to the function of Bik1p in the dynein pathway or to defects in spindle position. Furthermore, a sfi1-1 Deltakip2 mutant was viable, suggesting that the Bik1p pool at the cytoplasmic microtubule plus-ends may not be required in sfi1-1. Microscopic examination indicated the sfi1-1 mutant was delayed in SPB duplication, SPB separation, or spindle elongation and the sfi-1 Deltabik1 double mutant arrested with duplicated but unseparated SPBs. These results suggest that Bik1p has a previously uncharacterized function in the separation of duplicated SPBs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Príons/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/genética , Alelos , Dineínas/genética , Deleção de Genes , Genes Letais , Proteínas Associadas aos Microtúbulos/genética , Proteínas Motores Moleculares , Organismos Geneticamente Modificados , Príons/metabolismo , Saccharomyces cerevisiae/genética , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Distribuição Tecidual , Transcrição GênicaRESUMO
Translocation through nuclear pore complexes (NPCs) requires interactions between receptor-cargo complexes and phenylalanine-glycine (FG) repeats in multiple FG domain-containing NPC proteins (FG-Nups). We have systematically deleted the FG domains of 11 Saccharomyces cerevisiae FG-Nups in various combinations. All five asymmetrically localized FG domains deleted together were non-essential. However, specific combinations of symmetrically localized FG domains were essential. Over half the total mass of FG domains could be deleted without loss of viability or the NPC's normal permeability barrier. Significantly, symmetric deletions caused mild reductions in Kap95-Kap60-mediated import rates, but virtually abolished Kap104 import. These results suggest the existence of multiple translocation pathways.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Poro Nuclear/metabolismo , Transporte Proteico/genética , Sequências Repetitivas de Aminoácidos/fisiologia , Saccharomyces cerevisiae/metabolismo , Permeabilidade da Membrana Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Deleção de Genes , Carioferinas/genética , Carioferinas/metabolismo , Mutação/genética , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Estrutura Terciária de Proteína/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
The interaction between nuclear pore proteins (nucleoporins) and transport factors is crucial for the translocation of macromolecules through nuclear pores. Many nucleoporins contain FG sequence repeats, and previous studies have demonstrated interactions between repeats containing FxFG or GLFG cores and transport factors. The crystal structure of residues 1-442 of importin-beta bound to a GLFG peptide indicates that this repeat core binds to the same primary site as FxFG cores. Importin-beta-I178D shows reduced binding to both FxFG and GLFG repeats, consistent with both binding to an overlapping site in the hydrophobic groove between the A-helices of HEAT repeats 5 and 6. Moreover, FxFG repeats can displace importin-beta or its S. cerevisiae homologue, Kap95, bound to GLFG repeats. Addition of soluble GLFG repeats decreases the rate of nuclear protein import in digitonin-permeabilized HeLa cells, indicating that this interaction has a role in the translocation of carrier-cargo complexes through nuclear pores. The binding of GLFG and FxFG repeats to overlapping sites on importin-beta indicates that functional differences between different repeats probably arise from differences in their spatial organization.
