SPINDLE POLE BODY PHOSPHOPROTEOME

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。 在2005年7月发表在Journal of Cell Biology上的论文中，Mike Rout的实验室（Rockefeller）报道了惊人的发现，即他们可以使用蛋白A标记的Mlp 2在IgG磁珠上分离完整的SPB（Niepel等人（2005）J. Cell Biol.170：225-235）。SPB是酵母中心体。 MLP 2基因和相关的MLP 1基因编码与脊椎动物Tpr蛋白相关的丝状蛋白。 Mlp 1和Mlp 2附着在核孔复合物的核表面，但Mlp 2也通过直接结合核心组分Spc 110、Spc 42和Spc 29而接触SPBs。 我们建议使用标记的Mlp 2分离SPB的能力，着眼于识别SPB组分上的磷酸化位点。 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种核心SPB组分中的11种。 虽然这种技术显然有效，但它对于鉴定复合物中的所有蛋白质或鉴定磷酸化肽不是最佳的。 我们建议使用MuDPIT和其他优化用于识别磷酸化位点以创建SPB磷酸化蛋白质组的策略重新审视Mlp 2-SPBs的质谱分析。

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.