RESUMO
Here, we present a protocol for establishing a protein interactome based on close physical proximity to a target protein within live yeast cells. We describe steps for capturing both transient and stable binders by integrating a non-natural amino acid. We detail procedures for employing a site-directed method for labeling the surface that mediates protein associations and uncovers the binding sites on the interactors. Combined with mass spectrometry, our approach proves valuable in discovering binding partners and constructing a comprehensive protein-interaction network.
Assuntos
Saccharomycetales , Saccharomycetales/metabolismo , Proteínas/metabolismo , Sítios de Ligação , Mapas de Interação de Proteínas , Aminoácidos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Molecular chaperones govern proteome health to support cell homeostasis. An essential eukaryotic component of the chaperone system is Hsp90. Using a chemical-biology approach, we characterized the features driving the Hsp90 physical interactome. We found that Hsp90 associated with â¼20% of the yeast proteome using its three domains to preferentially target intrinsically disordered regions (IDRs) of client proteins. Hsp90 selectively utilized an IDR to regulate client activity as well as maintained IDR-protein health by preventing the transition to stress granules or P-bodies at physiological temperatures. We also discovered that Hsp90 controls the fidelity of ribosome initiation that triggers a heat shock response when disrupted. Our study provides insights into how this abundant molecular chaperone supports a dynamic and healthy native protein landscape.
Assuntos
Proteínas Intrinsicamente Desordenadas , Chaperonas Moleculares , Proteoma , Humanos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismoRESUMO
Chromosomes are selectively organized within the nuclei of interphase cells reflecting the current fate of each cell and are reorganized in response to various physiological cues to maintain homeostasis. Although substantial progress is being made to establish the various patterns of genome architecture, less is understood on how chromosome folding/positioning is achieved. Here, we discuss recent insights into the cellular mechanisms dictating chromatin movements including the use of epigenetic modifications and allosterically regulated transcription factors, as well as a nucleoskeleton system comprised of actin, myosin, and actin-binding proteins. Together, these nuclear factors help coordinate the positioning of both general and cell-specific genomic architectural features.
Assuntos
Células Eucarióticas/química , Genoma , Actinas/metabolismo , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromossomos/química , Cromossomos/metabolismo , Epigênese Genética , Humanos , Interfase , Miosinas/metabolismoRESUMO
Movement of chromosome sites within interphase cells is critical for numerous pathways including RNA transcription and genome organization. Yet, a mechanism for reorganizing chromatin in response to these events had not been reported. Here, we delineate a molecular chaperone-dependent pathway for relocating activated gene loci in yeast. Our presented data support a model in which a two-authentication system mobilizes a gene promoter through a dynamic network of polymeric nuclear actin. Transcription factor-dependent nucleation of a myosin motor propels the gene locus through the actin matrix, and fidelity of the actin association was ensured by ARP-containing chromatin remodelers. Motor activity of nuclear myosin was dependent on the Hsp90 chaperone. Hsp90 further contributed by biasing the remodeler-actin interaction toward nucleosomes with the non-canonical histone H2A.Z, thereby focusing the pathway on select sites such as transcriptionally active genes. Together, the system provides a rapid and effective means to broadly yet selectively mobilize chromatin sites.