RESUMEN
Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degradation. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degradation during recovery from shock, contrasting with the rapid degradation observed for many exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.
Asunto(s)
Respuesta al Choque Térmico , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Cicloheximida/farmacología , Gránulos Citoplasmáticos/metabolismo , Agregado de Proteínas , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Evolving lineages face a constant intracellular threat: most new coding sequence mutations destabilize the folding of the encoded protein. Misfolded proteins form insoluble aggregates and are hypothesized to be intrinsically cytotoxic. Here, we experimentally isolate a fitness cost caused by toxicity of misfolded proteins. We exclude other costs of protein misfolding, such as loss of functional protein or attenuation of growth-limiting protein synthesis resources, by comparing growth rates of budding yeast expressing folded or misfolded variants of a gratuitous protein, YFP, at equal levels. We quantify a fitness cost that increases with misfolded protein abundance, up to as much as a 3.2% growth rate reduction when misfolded YFP represents less than 0.1% of total cellular protein. Comparable experiments on variants of the yeast gene orotidine-5'-phosphate decarboxylase (URA3) produce similar results. Quantitative proteomic measurements reveal that, within the cell, misfolded YFP induces coordinated synthesis of interacting cytosolic chaperone proteins in the absence of a wider stress response, providing evidence for an evolved modular response to misfolded proteins in the cytosol. These results underscore the distinct and evolutionarily relevant molecular threat of protein misfolding, independent of protein function. Assuming that most misfolded proteins impose similar costs, yeast cells express almost all proteins at steady-state levels sufficient to expose their encoding genes to selection against misfolding, lending credibility to the recent suggestion that such selection imposes a global constraint on molecular evolution.
Asunto(s)
Citosol/química , Proteínas Fúngicas/química , Proteínas Bacterianas/química , Citosol/metabolismo , Evolución Molecular , Calor , Proteínas Luminiscentes/química , Chaperonas Moleculares/química , Desnaturalización Proteica , Pliegue de Proteína , Proteínas/química , Proteómica/métodos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Transcripción GenéticaRESUMEN
Rod-shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: the Rod complex moves around the cell circumference, whereas class A penicillin-binding proteins (aPBPs) do not. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each affects the growth and width of Bacillus subtilis as well as the mechanical anisotropy and orientation of material within their sacculi. Rod width is not determined by MreB, rather it depends on the balance between the systems: the Rod complex reduces diameter, whereas aPBPs increase it. Increased Rod-complex activity correlates with an increased density of directional MreB filaments and a greater fraction of directional PBP2a enzymes. This increased circumferential synthesis increases the relative quantity of oriented material within the sacculi, making them more resistant to stretching across their width, thereby reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems builds and maintains rods of different widths. Escherichia coli Rod mutants also show the same correlation between width and directional MreB filament density, suggesting this model may be generalizable to bacteria that elongate via the Rod complex.
Asunto(s)
Bacillus subtilis/citología , Bacillus subtilis/metabolismo , Pared Celular/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Unión a las Penicilinas/metabolismoRESUMEN
Neuromodulatory cells transduce environmental information into long-lasting behavioral responses. However, the mechanisms governing how neuronal cells influence behavioral plasticity are difficult to characterize. Here, we adapted the translating ribosome affinity purification (TRAP) approach in C. elegans to profile ribosome-associated mRNAs from three major tissues and the neuromodulatory dopaminergic and serotonergic cells. We identified elc-2, an Elongin C ortholog, specifically expressed in stress-sensing amphid neuron dual ciliated sensory ending (ADF) serotonergic sensory neurons, and we found that it plays a role in mediating a long-lasting change in serotonin-dependent feeding behavior induced by heat stress. We demonstrate that ELC-2 and the von Hippel-Lindau protein VHL-1, components of an Elongin-Cullin-SOCS box (ECS) E3 ubiquitin ligase, modulate this behavior after experiencing stress. Also, heat stress induces a transient redistribution of ELC-2, becoming more nuclearly enriched. Together, our results demonstrate dynamic regulation of an E3 ligase and a role for an ECS complex in neuromodulation and control of lasting behavioral states.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Cullin/genética , Elonguina/genética , Genoma , Proteínas Supresoras de la Señalización de Citocinas/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cullin/metabolismo , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Elonguina/metabolismo , Conducta Alimentaria/fisiología , Edición Génica , Regulación de la Expresión Génica , Respuesta al Choque Térmico/genética , Calor , Humanos , Larva/genética , Larva/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Neuronas Serotoninérgicas/citología , Neuronas Serotoninérgicas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Countless studies monitor the growth rate of microbial populations as a measure of fitness. However, an enormous gap separates growth-rate differences measurable in the laboratory from those that natural selection can distinguish efficiently. Taking advantage of the recent discovery that transcript and protein levels in budding yeast closely track growth rate, we explore the possibility that growth rate can be more sensitively inferred by monitoring the proteomic response to growth, rather than growth itself. We find a set of proteins whose levels, in aggregate, enable prediction of growth rate to a higher precision than direct measurements. However, we find little overlap between these proteins and those that closely track growth rate in other studies. These results suggest that, in yeast, the pathways that set the pace of cell division can differ depending on the growth-altering stimulus. Still, with proper validation, protein measurements can provide high-precision growth estimates that allow extension of phenotypic growth-based assays closer to the limits of evolutionary selection.
Asunto(s)
Proteínas Fúngicas/metabolismo , Aptitud Genética/fisiología , Saccharomycetales/crecimiento & desarrollo , Transcriptoma/fisiología , Funciones de Verosimilitud , Proteómica , Saccharomycetales/metabolismo , Selección Genética , Transcriptoma/genéticaRESUMEN
Chromatin plays roles in processes governed by different time scales. To assay the dynamic behavior of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested Saccharomyces cerevisiae at single-nucleosome resolution over 4% of the genome, and at lower (approximately 265 base pair) resolution over the entire genome. We find that nucleosomes at promoters are replaced more rapidly than at coding regions and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states.
Asunto(s)
Genoma Fúngico , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Replicación del ADN , Fase G1 , Genes Fúngicos , Cinética , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/citología , Sitio de Iniciación de la TranscripciónRESUMEN
The histone code hypothesis holds that covalent posttranslational modifications of histone tails are interpreted by the cell to yield a rich combinatorial transcriptional output. This hypothesis has been the subject of active debate in the literature. Here, we investigated the combinatorial complexity of the acetylation code at the four lysine residues of the histone H4 tail in budding yeast. We constructed yeast strains carrying all 15 possible combinations of mutations among lysines 5, 8, 12, and 16 to arginine in the histone H4 tail, mimicking positively charged, unacetylated lysine states, and characterized the resulting genome-wide changes in gene expression by using DNA microarrays. Only the lysine 16 mutation had specific transcriptional consequences independent of the mutational state of the other lysines (affecting approximately 100 genes). In contrast, for lysines 5, 8, and 12, expression changes were due to nonspecific, cumulative effects seen as increased transcription correlating with an increase in the total number of mutations (affecting approximately 1,200 genes). Thus, acetylation of histone H4 is interpreted by two mechanisms: a specific mechanism for lysine 16 and a nonspecific, cumulative mechanism for lysines 5, 8, and 12.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Genómica , Histonas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetilación , Cromosomas Fúngicos/genética , Perfilación de la Expresión Génica , Histonas/química , Histonas/genética , Lisina/genética , Familia de Multigenes/genética , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética/genéticaRESUMEN
The positioning of nucleosomes along chromatin has been implicated in the regulation of gene expression in eukaryotic cells, because packaging DNA into nucleosomes affects sequence accessibility. We developed a tiled microarray approach to identify at high resolution the translational positions of 2278 nucleosomes over 482 kilobases of Saccharomyces cerevisiae DNA, including almost all of chromosome III and 223 additional regulatory regions. The majority of the nucleosomes identified were well-positioned. We found a stereotyped chromatin organization at Pol II promoters consisting of a nucleosome-free region approximately 200 base pairs upstream of the start codon flanked on both sides by positioned nucleosomes. The nucleosome-free sequences were evolutionarily conserved and were enriched in poly-deoxyadenosine or poly-deoxythymidine sequences. Most occupied transcription factor binding motifs were devoid of nucleosomes, strongly suggesting that nucleosome positioning is a global determinant of transcription factor access.