The Novel Function Research Of Ssz1 Protein In Saccharomyces Cerevisiae

Objective: Overexpression of SSZ1 can suppress the temperature-sensitive phenotype of scj1-1Δire1 and rescue the synthetic lethal Δscj1Δire1. These phenomena indicate that SSZ1 may possess a cellular function different from those reported in previous papers in the case of its overexpression. In order to explore the novel features, we analyzed the transcriptional profiling of SSZ1 overexpressed in yeasts to predict its novel functions confirmed using further experiments. Additionally, we investigated the novel functions affected by the loss of SSZ1 structural domains. This research may provide a deep insight into understanding the SCJ1 function and the UPR mechanism.Methods: Firstly the primers of SSZ1 mutant were designed by Primer premier 5.0, and then the seven fragments were amplified from YCp50-TDH3-SSZ1 vector and inserted into vector YCp50 respectively by PCR. After identification by double-enzyme digestion and sequencing,the plasmids with SSZ1 mutants were transformed into widetype and temperature-sensitive yeasts. Serial dilution culture assay was carried out to analyze their effect on resistance to cycloheximide and the temperature-sensitive phenotype of the yeast scj1-1Δire1. The plasmids with SSZ1 mutants were also transformed into the yeast(Δscj1,ire1::IRE1,ade2,ade3::ADE3).Scribing method was used to verify the functions of SSZ1 mutants and the online software SWISS-MODEL was used to predict their protein structural products. Application of DNA microarray technology was used to predict the cellular functions of overexpression of SSZ1.Differentially expressed genes were analyzed by GO enrichment analysis and PPI network and then their authenticity was verified through biochemical experiments. The vectors of differentially expressed genes were constructed, and the serial dilution culture assay was carried out to analyze their cellular functions. Finally, the relationship between SSZ1 and three pathways PDR, UPR, CWI were confirmed through the analysis of variation on different expressed genes related with these pathways.Results:(1) SSZ1 deletion mutants were successfully constructed;(2) SSZ1 mutant plasmid(SSZ1Δ2-40, Δ2-115, Δ2-200, Δ2-240, Δ2-303, Δ2-372,Δ2-402) was transformed into wild-type yeast which cannot resist the cycloheximide;(3) The temperature-sensitive yeast(scj1-1Δire1)which was respectively transformed with SSZ1 mutant plasmid(SSZ1Δ2-200, Δ2-240, Δ2-372,Δ2-402) could grow well at 37 ℃ and this phenomenon was opposite to that in yeast (scj1-1Δire1) which was respectively transformed with SSZ1 mutant plasmids(SSZ1Δ2-40,Δ2-115, Δ2-303).(4) Color separation experiments showed that the SSZ1 mutants(SSZ1Δ2-200,Δ2-240, Δ2-372, Δ2-402) had the same functional phenotypes compared with SSZ1 of full length, and were different from SSZ1 mutant(SSZ1Δ2-40, Δ2-115, Δ2-303).(5) Transcriptional expression profiling showed that overexpression of SSZ1 could induce transcription of PDR-related genes and could not increase the transcriptional level of UPR-related genes.Moreover, overexpression of SSZ1 can up-regulate CWI-related genes and RSC30.(6) A number of differentially expressed genes were constructed into vectors and verified by sequencing. PDR1, PDR5, RHO1, SMP1 and KDX1 were respectively transformed into yeast(scj1-1Δire1) and the results showed that they cannot suppress the temperature-sensitive phenotype. However, the temperature-sensitive phenotype of yeast(scj1-1Δire1) can be suppressed with 1 M sorbitol. Furthermore, RSC30 suppressed the temperature-sensitive phenotype of scj1-1Δire1.Conclusion: Overexpression of SSZ1 can maintain the cellular robustness of yeasts. Ssz1 p domains are closely related to their new features, The amino acids from 403 to 538 in Ssz1 p are necessary for its new function. In addition, there are two domains(2-40, 241-303) for keeping their new features and another two domains(116-200,304-372) for inhibiting; Overexpression of SSZ1 can improve the integrity of the cell wall but it is not executed by PDR and UPR pathway. However, the new function does not depend on CWI pathway; Finally, we found that overexpression of RSC30 can inhibit temperature-sensitive phenotype of yeast(scj1-1Δire1),which suggested that the new features of Ssz1 p may be related to RSC30.