ROLE OF THE F-BOX PROTEIN POF6 IN CELL CYCLE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 色葡萄粟酒裂殖酵母F-box蛋白Pof 6是细胞活力所必需的，并且在其氨基末端部分含有F-box，在羧基末端部分含有两个Sec 10结构域，并且在终止密码子之前含有CAAX结构域。其最接近的同源物是来自S的Rcy 1。酿酒酵母（24%同一性），一种参与蛋白质再循环的非必需F盒蛋白（其还含有Sec 10结构域和CAAX结构域）。 Pof 6已被证明与Skp 1（F-box-binding adaptor protein）相互作用，并且大多数对Skp 1/F-box结构域界面重要的残基在Pof 6的F-box中是保守的。令人惊讶的是，与其他F-box蛋白不同，Pof 6被发现不与支架蛋白Cul 1相互作用，这提高了该F-box蛋白是非SCF复合物的一部分的可能性。这一假设得到了以下事实的支持：Cul 1/F-box结构域界面的重要残基在Pof 6中不保守。 为了破译Pof 6在细胞中的重要作用，与Dr. J. Thompson和Dr. J. Yates（TSRI，San Diego）合作进行TAP纯化，然后进行MudPIT分析。在几个新的Pof 6相互作用，高分子量的蛋白质，纯化的银染样品上可见，以及通过质谱检测（139独特的肽/792总肽）进行了鉴定。编码该蛋白的基因在S. pombe及其与Pof 6的相互作用已经通过免疫共沉淀证实。这种蛋白质的功能正在研究中。 ts pof 6突变体pof 6 -1166和pof 6 -51的表型表征揭示了两种菌株的相似缺陷：具有分隔缺陷的小尺寸细胞和含有4C DNA含量的细胞的积累。这些表型表明DNA复制不受影响，突变细胞在细胞生长而不是细胞周期进程中有缺陷。总而言之，迄今为止收集的数据使我们假设Pof 6功能与翻译机制有关，因为它似乎始终与核糖体组分相互作用，因此其功能障碍将导致生长缺陷。结果，两种pof 6 ts菌株似乎对翻译抑制剂敏感，支持Pof 6直接调节核糖体的生物发生或活性的观点。 随后的多聚核糖体分析（蔗糖梯度离心）表明，Pof 6与活性多聚核糖体，80 S和60 S复合物共沉淀。Skp 1的一部分与Pof 6共迁移，尽管大部分蛋白质与较小分子量的复合物（即SCF复合物）一起保留。Pof 6的失活极大地改变了多核糖体谱：翻译的多核糖体退化，似乎积累成无活性的单核糖体，导致80 S峰的增加。这个数字是翻译活动减少的特征。我们现在正计划使用多核糖体分析来检查Pof 6相互作用子是否与Pof 6和Skp 1在高分子量复合物处共沉积，并调查Pof 6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.