RESUMO
Macromolecular complexes are responsible for most of the essential mechanisms in cells, leading to a broad interest in their purification and characterization. Co-expression is now widely recognized as a major technique for assembling multiprotein complexes and many co-expression systems are currently available for performing co-expression experiments in different hosts. However, comparative knowledge on co-expression strategies is still crucially lacking. Using versatile co-expression systems for Escherichia coli, the pET-MCN and pET-MCP series, and ternary protein complexes as examples, we demonstrate how to successfully delineate correct co-expression strategies. Specifically, an appropriate, complex-dependent approach alleviates stoichiometry imbalance and yield problems, and even failure in producing complexes. Importantly, some of the parameters influencing co-expression strategies appear independent of the expression host, thus having implications for co-expression in eukaryotic hosts. By further using these strategies, we show that co-expression in E. coli enables reconstitution of protein complexes as large as the deubiquitination module of the SAGA transcription factor and the histone octamer.
Assuntos
Clonagem Molecular/métodos , Histonas/genética , Complexos Multiproteicos/genética , Proteínas Recombinantes/genética , Animais , Fator de Ligação a CCAAT/biossíntese , Fator de Ligação a CCAAT/química , Fator de Ligação a CCAAT/genética , Drosophila , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Escherichia coli/genética , Vetores Genéticos , Histonas/biossíntese , Humanos , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/química , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/biossíntese , Transativadores/química , Transativadores/genéticaRESUMO
Mitosis ensures equal segregation of the genome and is controlled by a variety of ubiquitylation signals on substrate proteins. However, it remains unexplored how the versatile ubiquitin code is read out during mitotic progression. Here, we identify the ubiquitin receptor protein UBASH3B as an important regulator of mitosis. UBASH3B interacts with ubiquitylated Aurora B, one of the main kinases regulating chromosome segregation, and controls its subcellular localization but not protein levels. UBASH3B is a limiting factor in this pathway and is sufficient to localize Aurora B to microtubules prior to anaphase. Importantly, targeting Aurora B to microtubules by UBASH3B is necessary for the timing and fidelity of chromosome segregation in human cells. Our findings uncover an important mechanism defining how ubiquitin attachment to a substrate protein is decoded during mitosis.
Assuntos
Aurora Quinase B/metabolismo , Segregação de Cromossomos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Ubiquitina/metabolismo , Anáfase/fisiologia , Linhagem Celular , Células HeLa , Humanos , Cinetocoros/metabolismo , Fosforilação , Ubiquitinação/fisiologiaRESUMO
Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22. (1) In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas Culina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Especificidade por Substrato , Ubiquitinação , Quinase 1 Polo-LikeRESUMO
Polo-like kinase 1 (PLK1) critically regulates mitosis through its dynamic localization to kinetochores, centrosomes and the midzone. The polo-box domain (PBD) and activity of PLK1 mediate its recruitment to mitotic structures, but the mechanisms regulating PLK1 dynamics remain poorly understood. Here, we identify PLK1 as a target of the cullin 3 (CUL3)-based E3 ubiquitin ligase, containing the BTB adaptor KLHL22, which regulates chromosome alignment and PLK1 kinetochore localization but not PLK1 stability. In the absence of KLHL22, PLK1 accumulates on kinetochores, resulting in activation of the spindle assembly checkpoint (SAC). CUL3-KLHL22 ubiquitylates Lys 492, located within the PBD, leading to PLK1 dissociation from kinetochore phosphoreceptors. Expression of a non-ubiquitylatable PLK1-K492R mutant phenocopies inactivation of CUL3-KLHL22. KLHL22 associates with the mitotic spindle and its interaction with PLK1 increases on chromosome bi-orientation. Our data suggest that CUL3-KLHL22-mediated ubiquitylation signals degradation-independent removal of PLK1 from kinetochores and SAC satisfaction, which are required for faithful mitosis.