Preliminary Study On The Involvement Of Spc29p Phosphorylation In Regulating The Interaction Between N-Sfi1p And Spc29p Of Saccharomyces Cerevisiae

In the yeast Saccharomyces cerevisiae cells, the spindle pole body (SPB) acts asthe microtubule organizing center and plays an important role in cell division andgenome stability. It shares a number of homologous structural components andregulators with the centrosome, which is microtubule organizing center in themammalian cells, and some parallels in the process of their duplication and separationcan also be seen, which makes the yeast SPB a useful model for studying theprinciples and mechanisms governing centrosome assembly and separation.SPB consists of two substructures, the core SPB and the half bridge. Sfi1p is anessential component of SPB, which localizes at half bridge and spans its full length.Sfi1p, whose deletion or mutation causes a failure of SPB replication, is required forSPB duplication and self-assembly. A large number of studies show that Sfi1p has itshomologous protein hSfi1p in the mammalian centrosome. Spc29p is a necessarycoiled-coil element of central plaque, a member of the core SPB components.Temperature sensitive (ts) alleles of SPC29will lead to a defect in SPB duplication atrestrictive temperature. Previous studies in our lab show that Spc29p interact with theN terminus of Sfi1p, and the strong interaction plays an essential role in early SPBduplication, providing a direct link between the core SPB and the half bridge for thefirst time. Spc29p is phosphorylated on four sites, namely T18, T159, S187and T240,by the Mps1p kinase. In this work, we observed the involvement of Spc29pphosphorylation in regulating the interaction between Spc29p and N-Sfi1p. We alsoelucidated the function of Spc29p phosphorylation in SPB self-assembly. The studywill not only provide great help to solve important scientific problems in the SPBduplication, but also lay a foundation for investigation of the mammalian centrosome.We constructed phospho-abolishing plasmids pGAD424-spc29-T18A,-T159A,-S187A and-4A, phospho-mimicking plasmids pGAD424-spc29-T18D,-T159D,-S187D and-4D by fusion PCR, site-directed mutagenesis techniques and othercloning means. We got the correct above plasmids by DNA and protein sequencesblast between spc29allele and SPC29WT. Moreover, having plasmids of pGAD424,pGBT9， pGAD424-SPC29， pGBT9-SPC29， pGBT9-SFI1-NT(1-250),pGAD424-spc29-T240A，and-T240D in our lab, we next tested interactions of the N terminus of SFI1with the SPC29or spc29alleles in a two-hybrid assay. Thistwo-hybrid assay clearly showed that the phospho-mimicking spc29-4D alleledrastically reduced its interaction with the N terminus of SFI1comparing tophospho-abolishing spc29-4A allele, which meant Spc29p phosphorylation downregulated the affinity of Spc29p for N-Sfi1p. Specifically, phosphorylation of Spc29pweakened the interaction between Spc29p and N-Sfi1p, and then the associationbetween the core SPB and the half bridge was affected. Spc29p phosphorylation mayconstitutes a novel mechanism for regulation of SPB duplication in the yeast.