ROLE OF THE F-BOX PROTEIN POF6 IN CELL CYCLE AND TRANSCRIPTION REGULATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Pombe F-box蛋白POF6是细胞存活所必需的，在其氨基末端含有一个F-box，在羧基末端含有两个Sec10结构域，在终止密码子之前含有一个CAAX结构域。它最接近的同源物是酿酒酵母的Rcy1(同源性为24%)，这是一种参与蛋白质循环的非必需F-box蛋白(它还包含一个Sec10结构域和一个CAAX结构域)。 POF6已被证明与Skp1(F-box结合接头蛋白)持续相互作用，并且大多数对Skp1/F-box结构域界面重要的残基在POF6的F-box中保守。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，这增加了该F-box蛋白是非SCF复合体的可能性。这一假说得到了以下事实的支持，即在POF6中，对Cul1/F-box结构域界面重要的残基不是保守的。 为了破译POF6在细胞中的重要作用，与圣地亚哥TSRI的J.Thompson博士和J.Yates博士合作进行了TAP纯化和MudPIT分析。在几个新的Pof6相互作用子中，鉴定出一种高相对分子质量的蛋白质，在纯化的银染样品上可见，并能很好地被质谱仪检测到(139个独特的多肽/792个总多肽)。编码该蛋白的基因在S.pombe中是必不可少的，它与POF6的相互作用已经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的表型特征表明，这两个菌株存在相似的缺陷：具有分隔缺陷的小细胞和含有4C DNA含量的细胞积累。这些表型表明DNA复制没有受到影响，突变细胞在细胞生长方面存在缺陷，而不是细胞周期进展。综上所述，到目前为止收集的数据使我们假设Pof6的功能与翻译机制有关，因为它似乎一直与核糖体成分相互作用，因此其功能障碍将导致生长缺陷。结果，两株POF6TS菌株似乎对翻译抑制剂都很敏感，这支持了POF6直接调节核糖体的生物发生或活性的观点。 随后的多聚体图谱分析(蔗糖梯度离心法)显示，POF6与活性多聚体、80S和60S复合体共沉淀。Skp1的一部分与POF6共迁移，尽管大部分蛋白质仍然以较小的分子量复合体(即SCF复合体)存在。POF6的失活极大地改变了多聚体的分布：翻译的多聚体退化，似乎积累成不活跃的单核糖体，导致80s峰的增加。这一数字是翻译活动减少的特征。我们现在正计划使用多聚体图谱分析来检查POF6相互作用体是否与POF6和Skp1在高分子量复合体上共沉淀，并调查POF6是否参与翻译起始和/或延伸。

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.