ROLE OF THE F-BOX PROTEIN POF6 IN CELL CYCLE

F-BOX 蛋白 POF6 在细胞周期中的作用

• 批准号: 7957672
• 负责人: TAKASHI TODA
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957672
• 关键词: Adaptor Signaling Protein; Affect; Binding; Biogenesis; Biology; Cell Cycle; Cell Cycle Progression; Cell Size; Cell Survival; Cells; Centrifugation; Characteristics; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA biosynthesis; Data; Defect; F Box Domain; F-Box Proteins; Fission Yeast; Functional disorder; Funding; Fungal Genome; Genes; Grant; Growth; Homologous Gene; Institution; Investigation; Lead; Mass Spectrum Analysis; Molecular Weight; Mono-S; Peptides; Phenotype; Ploidies; Polyribosomes; Proteins; Recycling; Research; Research Personnel; Resources; Ribosomes; Role; Saccharomyces cerevisiae; Sampling; Scaffolding Protein; Silver Staining; Source; Sucrose; Terminator Codon; Translating; Translation Initiation; Translations; United States National Institutes of Health; cell growth; inhibitor/antagonist; mutant; protein function

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The S. pombe F-box protein Pof6 is essential for cell viability and contains an F-box in its amino-terminal portion, two Sec10 domains in the carboxy-terminal portion and a CAAX domain prior to the stop codon. Its closest homologue is Rcy1 from S. cerevisiae (24% identity), a non-essential F-box protein involved in protein recycling (which also contains a Sec10 domain and a CAAX domain). Pof6 has been shown to interact with Skp1 (the F-box-binding adaptor protein) consistently and, most of the residues important for the Skp1/F-box domain interface are conserved in the F-box of Pof6. Surprisingly, unlike other F-box proteins, Pof6 was found not to interact with the scaffold protein Cul1 raising the possibility that this F-box protein is part of a non-SCF complex. This hypothesis is supported by the fact that the residues important for the Cul1/F-box domain interface are not conserved in Pof6. To decipher the essential role for Pof6 in the cell, a TAP purification followed by MudPIT analysis was performed in collaboration with Dr. J. Thompson and Dr J. Yates, TSRI, San Diego. Among several new Pof6 interactors, a protein of high molecular weight, visible on purified silver-stained samples and well detected by mass spectrometry (139 unique peptides/792 total peptides) was identified. The gene encoding for this protein is essential in S. pombe and its interaction with Pof6 has been confirmed by coimmunoprecipitation. The function of this protein is now under investigation. The phenotypic characterization of the ts pof6 mutants, pof6-1166 and pof6-51 reveals similar defects for both strains: small sized cells with septation defects and an accumulation of cells containing 4C DNA content. These phenotypes suggest that DNA replication is not affected and that mutant cells are defective in cell growth rather than cell cycle progression. Altogether, the data collected so far lead us to the hypothesis that Pof6 function is related to the translation machinery as it seems to consistently interact with ribosome components, therefore its dysfunction would result in growth defects. As a result both pof6 ts strains appear to be sensitive to translation inhibitors, supporting the idea that Pof6 directly modulates the biogenesis or the activity of the ribosomes. Subsequently polysome profiling analysis (sucrose gradient centrifugation) showed that Pof6 co-sediments with the active polysomes, the 80S and 60S complexes. A portion of Skp1 co-migrates with Pof6, although most of the protein remains with smaller molecular weight complexes (i.e. SCF complexes). Inactivation of Pof6 modifies dramatically the polysome profile: the translating polysomes regressed and seem to accumulate into inactive mono-ribosomes, resulting in the increase of the 80S peak. This figure is characteristic of a diminution in translation activity. We are now planning to use the polysome profiling analysis to examine whether the Pof6 interactor co-sediments with Pof6 and Skp1 at the high molecular weight complexes, and investigate if Pof6 is involved in translation initiation and/or elongation.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 粟酒裂殖酵母 F-box 蛋白 Pof6 对于细胞活力至关重要，其氨基末端部分包含一个 F-box，羧基末端部分包含两个 Sec10 结构域，终止密码子之前包含一个 CAAX 结构域。其最接近的同源物是来自酿酒酵母的 Rcy1（24% 同一性），一种参与蛋白质回收的非必需 F-box 蛋白（还包含 Sec10 结构域和 CAAX 结构域）。 Pof6 已被证明与 Skp1（F-box 结合接头蛋白）一致地相互作用，并且大多数对 Skp1/F-box 结构域接口重要的残基都保守在 Pof6 的 F-box 中。令人惊讶的是，与其他 F-box 蛋白不同，Pof6 被发现不与支架蛋白 Cul1 相互作用，这提高了该 F-box 蛋白是非 SCF 复合物一部分的可能性。这一假设得到以下事实的支持：对于 Cul1/F-box 结构域接口重要的残基在 Pof6 中并不保守。 为了破译 Pof6 在细胞中的重要作用，我们与圣地亚哥 TSRI 的 J. Thompson 博士和 J. Yates 博士合作进行了 TAP 纯化，然后进行 MudPIT 分析。在几个新的 Pof6 相互作用蛋白中，鉴定出了一种高分子量蛋白质，在纯化的银染样品上可见，并且可以通过质谱法很好地检测到（139 个独特的肽/792 个总肽）。编码该蛋白的基因在粟酒裂殖酵母中是必需的，并且其与 Pof6 的相互作用已通过免疫共沉淀得到证实。这种蛋白质的功能目前正在研究中。 ts pof6 突变体 pof6-1166 和 pof6-51 的表型特征揭示了两种菌株的相似缺陷：具有分隔缺陷的小细胞和含有 4C DNA 内容物的细胞积累。这些表型表明 DNA 复制不受影响，并且突变细胞在细胞生长而不是细胞周期进展方面存在缺陷。总而言之，迄今为止收集的数据使我们得出这样的假设：Pof6 功能与翻译机制有关，因为它似乎始终与核糖体成分相互作用，因此其功能障碍将导致生长缺陷。因此，两种 pof6 ts 菌株似乎都对翻译抑制剂敏感，支持了 Pof6 直接调节核糖体生物发生或活性的观点。 随后的多核糖体分析（蔗糖梯度离心）表明，Pof6 与活性多核糖体、80S 和 60S 复合物共沉积。 Skp1 的一部分与 Pof6 共同迁移，但大部分蛋白质仍保留较小分子量的复合物（即 SCF 复合物）。 Pof6 的失活极大地改变了多核糖体的特征：翻译多核糖体退化并似乎积累成无活性的单核糖体，导致 80S 峰增加。这一数字表明翻译活动有所减少。我们现在计划使用多核糖体分析来检查 Pof6 相互作用子是否与 Pof6 和 Skp1 在高分子量复合物上共沉积，并研究 Pof6 是否参与翻译起始和/或延伸。