SPINDLE POLE BODY PHOSPHOPROTEOME

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。 在2005年7月发表在《细胞生物学杂志》上的一篇论文中，Mike Rout的实验室(Rockefeller)报告了一个惊人的发现，即他们可以使用蛋白A标记的Mlp2(Niepel等人)在免疫球蛋白磁珠上分离出完整、完整的SPBS。(2005)J.Cell Biol.170：225-235)。SPB是酵母的中心体。MLP2基因和相关的MLP1基因编码与脊椎动物Tpr蛋白相关的丝状蛋白。Mlp1和Mlp2附着在核孔复合体的核面上，但Mlp2也通过直接结合核心成分Spc110、Spc42和Spc29与SPBS接触。我们建议使用Mlp2的标记版本来分离SPB，并着眼于识别SPB组件上的磷酸化位点。研究人员通过从SPB制剂中的蛋白质凝胶中切除条带，鉴定了18个核心SPB组分中的11个。虽然这项技术显然有效，但对于鉴定复合体中的所有蛋白质或磷酸化多肽来说，它并不是最佳的。我们建议重新回顾Mlp2-SPBS的质谱学分析，使用泥坑和其他优化的策略来识别磷酸化位点，以创建SPB光蛋白质组。

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. In a July 2005 paper in the Journal of Cell Biology, Mike Rout's laboratory (Rockefeller) reported the startling finding that they could isolate entire, intact SPBs on IgG-magnetic beads using Protein A-tagged Mlp2 (Niepel et al. (2005) J. Cell Biol. 170:225-235). The SPB is the yeast centrosome. The MLP2 gene and the related MLP1 gene encode filamentous proteins related to the vertebrate Tpr protein. Mlp1 and Mlp2 attach to nuclear face of nuclear pore complexes, but Mlp2 also contacts SPBs via direct binding to the core components Spc110, Spc42 and Spc29. We propose to use the ability to isolate SPBs using a tagged version of Mlp2 with an eye to identifying phosphorylation sites on SPB components. The investigators identified 11 of the 18 core SPB components by excising bands from a gel of the proteins in the SPB preparation. While this technique clearly works, it is not optimal for the identification of all of the proteins in the complex or for the identification of phosphorylated peptides. We propose to revisit the mass spectrometric analysis of Mlp2-SPBs using MuDPIT and other tactics optimized for the identification of phosphorylation sites to create a SPB phoshoproteome.