SPINDLE POLE BODY PHOSPHOPROTEOME

点击翻译按钮获取中文摘要

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.