A Preliminary Study Pombe Ribosomal Protein L32-1 And L32-2 Functions

The ribosomal protein RPL32of Fission yeast Schizosaccharomyces pombe was coded by two paralogs:rp132-1and rp132-2. Our lab’s earlier research found that the ribosomal protein RPL32-2acted as a potential transcriptional activator in the fission yeast Schizosaccharomyces pombe. In further study, we found that when cultured in nutrient rich medium, both of the rp132-1△cells and rp132-2△cells were flocculated, and confirmed that the flocculation is caused by some cell surface glycoproteins.To find the cell wall proteins which caused cell flocculation, the transcriptional level of some cell wall surface protein coding genes were analyzed by qRT-PCR in the wild type and flocculation strains rp132-1△and rp132-2△. The results showed that the transcriptional level of SPAC186.01, SPBC947.04, SPAC977.07c,fta5and gas1have increased, of which SPAC186.01, SPAC977.07c have increased considerably. We conjecture that one or several proteins coded by these genes are the cell surface adhesion molecule which caused flocculation. Then, we constructed over-expression of SPAC186.01, SPBC359.04c, SPAC977.07c,fta5and gas1gene strains respectively. And found there was no flocculation formed in these five over-expression strains. At the same time, we found that cell flocculation has not been eliminated when deleted SPAC186.01, SPAC977.07c or gas1gene of flocculation strain rp132-1△. Thus, we speculate that the cell flocculation that caused by ribosomal gene deletion may not be caused by a single gene, but results of interaction of multiple genes. The over expression of these genes is one of the necessary condition for flocculation. Cell flocculation may be caused by multiple cell surface proteins, which have great similarity on the structure and function. When we knock out one of them, the other flocculation factor can substitute its function. Therefore the flocculation has not been eliminated.For validate whether the cell flocculation that caused by decreased expression of ribosome proteins is unique to Schizosaccharomyces pombe, we selected the s. cerevisiae strain BY4742as a material, to verify whether this phenomenon is universal. First, when knocked out the ribosomal protein coding genes rpl15A and rps26B, the S. Cerevisiae didn’t form flocculation as S.pombe. Then, we constructed flocculation strains of S. cerevisiae by the use of site-directed mutagenesis techniques. The quantitative PCR analysis revealed that the expression level of ribosomal protein genes in flocculation strains did not decline. Meanwhile, we analyzed the expression of several ribosomal protein genes of S. cerevisiae in the sexual flocculation and found that its expression level has not decreased. On the contrary, part of the ribosomal protein gene expression level has increased. Taken these experimental results together, we can easily conclude that the cell flocculation that caused by decreased expression of ribosome proteins isn’t exist in S. cerevisiae and it is unique to Schizosaccharomyces pom.be.Our previous study found that RPL32-2has a potential role in transcriptional regulation, while RPL32-1has not. In order to further study the function of RPL32-1and RPL32-2, we have constructed the over-expression of rp132-1and rp132-2strains respectively. The results showed that over-expression of rp132-1strain grow faster than the wild-type strain when cultured in the EMM medium. A small amount of heterocyst appeared in the stationary period. A large number of1C cells were detected in over-expression of rp132-1strain by flow cytometry analysis. Heat shock experiment reveal that over-expression of rp132-1cells have better heat resistance. All of these indicate the functional differences between RPL32-1and RPL32-2. We screened Grsl(mitochondrial and cytoplasmic glycine-tRNA ligase)that may specifically bind to RPL32-1through Co-IP technology. We used yeast two-hybrid analysis to further confirm the interactions between Grsl and RPL32-1and RPL32-2. The results show that there doesn’t exist interaction between them. Due to the limitations of the yeast two-hybrid, we need to use other methods to further research. This study demonstrated that there are functional differences between the ribosomal proteins RPL32-1and RPL32-2. They can be combined with different target protein to exercise some specific function. The result is of great significance for the further research of the function of RPL32-1and RPL32-2.