SPINDLE POLE BODY PHOSPHOPROTEOME

纺锤体磷酸化蛋白质组

• 批准号: 8365899
• 负责人: MARK WINEY
• 金额: $ 2.65万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365899
• 关键词: Binding; Biology; Cells; Centrosome; Complex; Face; Funding; Fungal Genome; Gel; Genes; Goals; Grant; Immunoglobulin G; Laboratories; National Center for Research Resources; Nuclear; Nuclear Pore Complex; Phosphorylated Peptide; Phosphorylation Site; Preparation; Principal Investigator; Proteins; Reporting; Research; Research Infrastructure; Research Personnel; Resources; Source; Techniques; United States National Institutes of Health; Work; Yeasts; cost; magnetic beads; spindle pole body

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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 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（或酵母中心体）（Niepel等人（2005）J. Cell Biol.170：225-235）。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，目的是鉴定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。 我们建议重新进行质谱分析Mlp 2-SPBs的磷酸化位点的识别，以创建一个SPB phoshoproteome。