SPINDLE POLE BODY PHOSPHOPROTEOME

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 Mike Rout的实验室(洛克菲勒)报告了一个惊人的发现，他们可以使用蛋白A标记的Mlp2在免疫球蛋白磁珠上分离完整的SPBS(或酵母中心体)(Niepel等人。(2005)J.Cell Biol.170：225-235)。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，目的是确定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。我们建议重新审视Mlp2-SPBS的质谱学分析，以确定磷酸化位点，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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Mike Rout's laboratory (Rockefeller) reported the startling finding that they could isolate entire, intact SPBs (or yeast centrosomes) on IgG-magnetic beads using Protein A-tagged Mlp2 (Niepel et al. (2005) J. Cell Biol. 170:225-235). 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 a goal to identify 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 for the identification of phosphorylation sites to create a SPB phoshoproteome.