Functional Characterisation of the Spindle Pole Body Component Bbp1p

Schramm, Carolin (2001) Functional Characterisation of the Spindle Pole Body Component Bbp1p. PhD thesis, University of Glasgow.

Abstract

In this study, Bbplp is described as a novel SPB component. BBPl was first identified as a high gene dosage suppresser of mutants in SPC29 (S. Elliott, published in Schramm et al., 2000), a gene coding for a component of the central plaque. Bbplp was shown to localise to the SPB by fluorescence microscopy. In more detail, Bbplp is present at the central plaque periphery as revealed by immunolabelling microscopy. Here, Bbplp interacts with Spc29p as further indicated by two-hybrid experiments and in vitro binding (S. Elliott, published in Schramm et al., 2000). In addition, Bbplp interacts with the half-bridge component Karlp in the yeast two-hybrid system. Via these interactions, Bbplp may connect the central plaque to the half-bridge. High gene dosage of SPC34, coding for a SPB component of unknown function, and SPC42, coding for a component of the central plaque rescued specific mutant alleles of BBP1, indicating further interactions of Bbplp with SPB components. The known two-hybrid interaction between Bbplp and Bfrlp (Xue et al., 1996) was tested and repeated, but a direct interaction between the two proteins could not be established. Bfrlp is localised in the cytoplasm and might be enriched at the nuclear envelope, indicating a local proximity of Bfrlp to Bbplp. Recent studies showed, that Bfrlp might be part of the Scpl60p/Pablp complex which is found at polyribosomes (Lang and Fridovich-Keil, 2000). Furthermore, Bbplp was shown to form a complex with Mps2p. Mps2p was identified by MALDI-MS in the affinity precipitate of Bbplp-ProA. Mps2p co-precipitates with Bbplp-3HA by immunoprecipitation and vice versa, Bbplp co-precipitates with Mps2p-3HA. Both proteins also interact in the yeast two-hybrid system. High gene dosage of MPS2 can suppress all known mutant alleles of BBP1. However, BBP1 is a high gene dosage suppresser of mps2-2 and mps2-42, but not mps2-l. Like Bbplp, Mps2p is a component of the SPB which is localised at the central plaque periphery as shown by fluorescence microscopy and immunolabelling electron microscopy. Analysis of mutants in BBP1 at the restrictive temperature revealed that they are defective in late steps of SPB duplication, like the proper insertion of the duplication plaque and the subsequent formation of the inner plaque. Failure in SPB duplication resulted in spindle formation and chromosome segregation defects. Bbpl-1 cells displayed a typical G2 arrest. An almost identical phenotype was observed before for mutants in MPS2 (Winey et ai, 1991). The functional similarity of Bbplp and Mps2p led to the hypothesis that the Bbplp/Mps2p complex functions in SPB duplication. In addition, the growth defect of bbpl and mps2 cells can be rescued by the deletion of POM152, a gene coding for a nuclear pore component. Interestingly, deletion of POM 152 rescues mutants in NDC1 (Chial et al., 1998), which are also defective in the insertion of the duplication plaque (Winey et al., 1993). BBPl is an essential gene (Xue et al., 1996; this study), but cells lacking MPS2 are partially viable and can grow very slowly (Munoz-Centeno et al., 1999; this study). Cells lacking MPS2 display various abnormal phenotypes including multiple bud formation, unsuccessful chromosome segregation and abnormal SPB content. In a suppresser screen SMY2 was identified as unique high gene dosage suppresser of Deltamps2 cells. SMY2 is the homologue of the fission yeast MPD2 which is a high gene dosage suppresser of CDC7 (Cullen et al., 2000), the budding yeast homologue of CDC15. In budding yeast high gene dosage of SMY2 is known to rescue mutants in MY02 (Lillie and Brown, 1994), coding for the yeast class V unconventional myosin 2. In addition, the mps2-42 allele is rescued by high gene dosage of HSC82 coding for one of the two budding yeast HSP90 chaperones. Hsp90p was shown to be a core component of the drosophila centrosome and it is required at different stages in the centrosome cycle (Lange et al., 2000). In addition to the Bbplp/Mps2p complex another novel complex consisting of Spc24p, Spc25p and NdcSOp was isolated (Janke et al., in press). Spc24p was enriched in precipitates of Ndc80p-6HA and Ndc80p co-precipitated with Spc24p-3HA. Furthermore, Spc24p co-precipitated with Spc25p-3HA. The direct interaction of the proteins was supported by the earlier finding that SPC24 interacts with SPC25 and NDC80 in in the yeast two-hybrid system (Cho et al., 1998). Recent studies in our laboratory showed that Spc24p, Spc25p and NdcSOp are part of a larger complex which associates with kinetochores (Janke et al, in press).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Elmar Schiebel
• Keywords: Cellular biology, Microbiology
• Date of Award: 2001
• Depositing User: Enlighten Team
• Unique ID: glathesis:2001-76029
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 17:06
• Last Modified: 19 Nov 2019 17:06
• URI: https://theses.gla.ac.uk/id/eprint/76029