Functional Characterization Of Sfi1Proteinin Saccharomyces Cerevisiae

As the microtubule organizing center of yeast cells, the spindle pole body (SPB) plays an essential role in cell division and maintenance of genome stability, and therefore has attracted a great deal of attention and enormous research efforts in elucidating its structure feature and function in the field of cell biology. Sfi1p is a newly identified SPB component of Saccharomyces cerevisiae, which localizes at Half-Bridge and spans its full length. Structural characterization and functional analysis of Sfi1p will not only shed new light on our current understanding about yeast SPB structure and function, but also aids investigation of its homologues, and hence the MTOC in higher eukaryotes. In this work, the role of Sfi1p, as a component of SPB, during mitosis, meiosis and karyogamy was investigated.Temperature sensitive (ts) alleles of SFI1were created by error-prone PCR. Every single mutation that resulted in the generation of the temperature sensitive alleles was identified by sequencing. The selected SFI1alleles with mutations localized within N-terminus, C-terminus or the middle region of the encoded protein were integrated individually into the SFI1locus of the haploid wild-type strain W303-1A, using a pop-in/pop-out gene replacement strategy.To explore possible genetic interactions between Sfi1p and the SPB components, genes encoding major components of the yeast SPB were individually overexpressed in yeast strains carrying SFI1temperature sensitive alleles. At restrictive temperature, SPC29could suppress Sfi1p N-terminal mutants, indicating that SFI1interacts genetically with SPC29. Interestingly, overexpression of SPC29also suppressed the temperature sensitive phenotype of sfi1-p3, a C-terminal truncated SFI1allele, probably via SPC110.In a two-hybrid assay, Spc29p was found to interact specifically with the N-terminus of Sfi1p and this is the only two hybrid interaction detected in our screen between Sfi1p and the major SPB components.In addition, Spc29-HA could be co-immunoprecipitated with Sfi1-MYC using an anti-MYC antibody, which was detected with an anti-HA antibody in immunobloting assay, demonstrating that Spc29p and Sfi1p could form a protein complex in vivo. Spc29p is known to be a phosphoprotein but the physiological role of Spc29p phosphorylation is not clear. In our co-immunoprecipitation assay we found that Sfi1p interacted mainly with the phosphorylated form of Spc29p. This result suggested that phosphorylation of Spc29p could be an important regulatory mechanism that controls SPB duplication, its structure integrity and dynamic function in SPB related cellular process.At restrictive temperature, it was observed that the fluorescence signal of Spc29-GFP decreased dramatically in some Sfi1p N-terminal mutants compared to wild-type strain, which indicated that the localization of Spc29-GFP was compromised in the mutant strains. These results suggested that the proper localization of Spc29p is dependent, at least partially, on the structural and functional integrity of Sfi1p in Saccharomyces cerevisiae.We also investigated the sporulation efficiency and the distribution of genetic materials into meiotically-produced haploid spores of the diploid mutant strains homogenous for specific SFI1ts alleles. Interestingly, some C-terminal ts mutants showed severe defects in sporulation efficiency even at permissive temperature and a significant portion of the sporulated mutant cells gave rise to dyads, the asci containing only two spores. Dyads from mutants778D (sfi1-778/sfi1-778) and791D (sfi1-791/sfi1-791) were dissected and the mating-type of resulting spores was assayed. While all the dyads from778D and a portion of the dyads from791D contained diploid spores, some dyads from791D were non-sister dyads. This phenomenon demonstrated that, in addition to its characterized function in SPB duplication, Sfi1p may also play an important role in yeast sporulation and loss of this function renders yeast cells defects in either meiosis or tetrad formation or both.At non-restrictive temperature, we observed that some Sfi1p ts mutants displayed a significant defect in zygotes formation during mating. This phenomenon suggested that Sfi1p may also play an important role in karyogamy.