RESUMEN
Previous studies have suggested that phosphorylation of translation elongation factor 1A (eEF1A) can alter its function, and large-scale phospho-proteomic analyses in Saccharomyces cerevisiae have identified 14 eEF1A residues phosphorylated under various conditions. Here, a series of eEF1A mutations at these proposed sites were created and the effects on eEF1A activity were analyzed. The eEF1A-S53D and eEF1A-T430D phosphomimetic mutant strains were inviable, while corresponding alanine mutants survived but displayed defects in growth and protein synthesis. The activity of an eEF1A-S289D mutant was significantly reduced in the absence of the guanine nucleotide exchange factor eEF1Bα and could be restored by an exchange-deficient form of the protein, suggesting that eEF1Bα promotes eEF1A activity by a mechanism other than nucleotide exchange. Our data show that several of the phosphorylation sites identified by high-throughput analysis are critical for eEF1A function.
Asunto(s)
Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis Mutacional de ADN , Fosforilación , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
We recently demonstrated that mammalian cells harbor nicotinamide adenine dinucleotide (NAD)-capped messenger RNAs that are hydrolyzed by the DXO deNADding enzyme. Here, we report that the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targeting different RNAs. The crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 Å resolution provides insights into the molecular basis of the deNADding activity in the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells, and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to nutrient or environmental stress manifests changes in NAD-capped RNA levels that are selectively responsive to Nudt12 or DXO, respectively, indicating an association of deNADding to cellular metabolism.
Asunto(s)
NAD/metabolismo , Pirofosfatasas/metabolismo , ARN Mensajero/metabolismo , Humanos , NAD/química , Pirofosfatasas/química , Pirofosfatasas/genética , ARN Mensajero/químicaRESUMEN
In most eukaryotic organisms, translation elongation requires two highly conserved elongation factors eEF1A and eEF2. Fungal systems are unique in requiring a third factor, the eukaryotic Elongation Factor 3 (eEF3). For decades, eEF3, a ribosome-dependent ATPase, was considered "fungal-specific", however, recent bioinformatics analysis indicates it may be more widely distributed among other unicellular eukaryotes. In order to determine whether divergent eEF3-like proteins from other eukaryotic organisms can provide the essential functions of eEF3 in budding yeast, the eEF3-like proteins from Schizosaccharomyes pombe and an oomycete, Phytophthora infestans, were cloned and expressed in Saccharomyces cerevisiae. Plasmid shuffling experiments showed that both S. pombe and P. infestans eEF3 can support the growth of S. cerevisiae in the absence of endogenous budding yeast eEF3. Consistent with its ability to provide the essential functions of eEF3, P. infestans eEF3 possessed ribosome-dependent ATPase activity. Yeast cells expressing P. infestans eEF3 displayed reduced protein synthesis due to defects in translation elongation/termination. Identification of eEF3 in divergent species will advance understanding of its function and the ribosome specific determinants that lead to its requirement as well as contribute to the identification of functional domains of eEF3 for potential drug discovery.
Asunto(s)
Factores de Elongación de Péptidos/metabolismo , Phytophthora infestans/metabolismo , Extensión de la Cadena Peptídica de Translación , Factores de Elongación de Péptidos/clasificación , FilogeniaRESUMEN
Protein synthesis requires factors that are proposed to enhance discrete steps. Eukaryotic initiation factor eIF5A was initially thought to affect initiation; however, it was later shown to facilitate translation elongation at polyproline. Recent work by Schuller et al. demonstrates that eIF5A facilitates both general elongation and termination in yeast, challenging these steps as silos.
Asunto(s)
Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.
Asunto(s)
Ciclo Celular , Toxina Diftérica/biosíntesis , Factor 2 Eucariótico de Iniciación/metabolismo , Expresión Génica , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Difosfato/genética , Adenosina Difosfato/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Toxina Diftérica/genética , Factor 2 Eucariótico de Iniciación/genética , Polirribosomas/genética , Polirribosomas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Eukaryotic translation elongation factor 1A (eEF1A) is one of the most abundant protein synthesis factors. eEF1A is responsible for the delivery of all aminoacyl-tRNAs to the ribosome, aside from initiator and selenocysteine tRNAs. In addition to its roles in polypeptide chain elongation, unique cellular and viral activities have been attributed to eEF1A in eukaryotes from yeast to plants and mammals. From preliminary, speculative associations to well characterized biochemical and biological interactions, it is clear that eEF1A, of all the translation factors, has been ascribed the most functions outside of protein synthesis. A mechanistic understanding of these non-canonical functions of eEF1A will shed light on many important biological questions, including viral-host interaction, subcellular organization, and the integration of key cellular pathways.
Asunto(s)
Factor 1 Eucariótico de Iniciación/química , Biosíntesis de Proteínas , ARN de Transferencia/química , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Selenocisteína/química , Actinas/química , Transporte Activo de Núcleo Celular , Apoptosis , Citoesqueleto/metabolismo , Factor 1 Eucariótico de Iniciación/metabolismo , Humanos , Factores de Elongación de Péptidos , Conformación Proteica , Virus ARN/metabolismoRESUMEN
The evolutionarily conserved PIF1 DNA helicase family is important for the maintenance of genome stability in the yeast, Saccharomyces cerevisiae. There are two PIF1 family helicases in S. cerevisiae, Pif1p and Rrm3p that both possess 5'-->3' DNA helicase activity but maintain unique functions in telomerase regulation and semi-conservative DNA replication. Database analysis shows that the PIF1 helicase family is represented by a single homologue in higher eukaryotes. To analyze the function of PIF1 homologues in mammals, we cloned the full length human PIF (hPIF) cDNA. Comparison of hPIF with its S. cerevisiae homologues showed that human PIF is equally similar to Pif1p and Rrm3p. Human PIF was expressed at low levels in a variety of tissues and immunofluorescence analysis showed that ectopic hPIF was localized to nuclear foci. hPIF was expressed in late S/G2 phase of the cell cycle and this cell cycle regulated abundance was conferred by both cell cycle regulated mRNA accumulation and ubiquitin-mediated degradation. Furthermore, hPIF is likely a target of the anaphase promoting complex/cyclosome as its abundance was decreased when an activator of the APC/C was overexpressed. Finally, antibodies against hPIF immunoprecipitated telomerase activity from human cell lines, and we have observed a physical interaction between hPIF and the catalytic subunit of telomerase, hTERT. Our data suggest that human PIF, like S. cerevisiae Pif1p, plays a role in telomerase regulation.
Asunto(s)
Ciclo Celular , ADN Helicasas/metabolismo , Telomerasa/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Núcleo Celular/metabolismo , Clonación Molecular , ADN Helicasas/química , ADN Helicasas/genética , Perfilación de la Expresión Génica , Células HeLa , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
The myc proto-oncogenes encode transcriptional regulators whose inappropriate expression is correlated with a wide array of human malignancies. Up-regulation of Myc enforces growth, antagonizes cell cycle withdrawal and differentiation, and in some situations promotes apoptosis. How these phenotypes are elicited is not well understood, largely because we lack a clear picture of the biologically relevant downstream effectors. We created a new biological system for the optimal profiling of Myc target genes based on a set of isogenic c-myc knockout and conditional cell lines. The ability to modulate Myc activity from essentially null to supraphysiological resulted in a significantly increased and reproducible yield of targets and revealed a large subset of genes that respond optimally to Myc in its physiological range of expression. The total extent of transcriptional changes that can be triggered by Myc is remarkable and involves thousands of genes. Although the majority of these effects are not direct, many of the indirect targets are likely to have important roles in mediating the elicited cellular phenotypes. Myc-activated functions are indicative of a physiological state geared toward the rapid utilization of carbon sources, the biosynthesis of precursors for macromolecular synthesis, and the accumulation of cellular mass. In contrast, the majority of Myc-repressed genes are involved in the interaction and communication of cells with their external environment, and several are known to possess antiproliferative or antimetastatic properties.
Asunto(s)
Perfilación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Línea Celular , Cicloheximida/farmacología , Prueba de Complementación Genética , Humanos , Cinética , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/genética , Transgenes/fisiologíaRESUMEN
The Saccharomyces cerevisiae Pif1p DNA helicase is the prototype member of a helicase subfamily conserved from yeast to humans. S. cerevisiae has two PIF1-like genes, PIF1 itself and RRM3, that have roles in maintenance of telomeric, ribosomal, and mitochondrial DNA. Here we describe the isolation and characterization of pfh1+, a Schizosaccharomyces pombe gene that encodes a Pif1-like protein. Pfh1p was the only S. pombe protein with high identity to Saccharomyces Pif1p. Unlike the two S. cerevisiae Pif1 subfamily proteins, the S. pombe Pfh1p was essential. Like Saccharomyces Pif1p, a truncated form of the S. pombe protein had 5' to 3' DNA helicase activity. Point mutations in an invariant lysine residue in the ATP binding pocket of Pfh1p had the same phenotype as deleting pfh1+, demonstrating that the ATPase/helicase activity of Pfh1p was essential. Although mutant spores depleted for Pfh1p proceeded through S phase, they arrested with a terminal cellular phenotype consistent with a postinitiation defect in DNA replication. Telomeric DNA was modestly shortened in the absence of Pfh1p. However, genetic analysis demonstrated that maintenance of telomeric DNA was not the sole essential function of S. pombe Pfh1p.