Study On The Interaction Between Coronin Protein And Septin Family Members In Yeast

Cell cycle is a fundamental process in maintaining cell activity,and its precise regulation plays an important role in cell survival,reproduction,development and heredity.Septin has a conserved gene family in yeast and human.The main components of Septin in Saccharomyces cerevisiae include Cdc3 p,Cdc10p,Cdc11 p,Cdc12p and Shs1p/Sep7 p.Structural alteration of Septin closely relates to cell cycle.The actin cytoskeleton is a complicate protein network that participates various physiological processes involved in including maintenance of cell shape,cell cycle,signal transduction,proliferation,differentiation and expressed gene regulation.Coronin protein(Crn1p)is a highly conserved protein that associated with actin skeletal regulation in eukaryotes.Crn1 p plays an important role in polymerization and depolymerization of actin cytoskeleton.However,the mechanisms of actin skeleton involves in cell cycle regulation have been rarely explored.In this work,we studied the interaction between Coronin protein and Cdc3 p,Cdc10p,Cdc11 p,Cdc12p and Shs1 p / Sep7 p in Saccharomyces cerevisiae by using Saccharomyces cerevisiae as the research material.The main results were listed as follows:(1)Interaction between Cdc11 p and Crn1 p was screened out by yeast two-hybrid assay.Using the genomic DNA from S288 c as the template,the full-length gene fragments of CDC10,CDC11 and CDC12 were amplified.The sizes were 969 bp,1248 bp and 1224 bp,respectively.The recombinant plasmids pGBKT7-CDC10,pGBKT7-CDC11 and pGBKT7-CDC12 were constructed.pGADT7-CRN1 was transformed into Y190 with the above three recombinant plasmids respectively.Interaction between Cdc11 p and Crn1 p was screened by the detection of ?-galactosidase activity.(2)Interaction between Crn1 p and Cdc11 p was confirmed by Pull-down assay.Using the pMD19-T-CDC11 as the template,the full-length gene fragment of CDC11 was amplified.The prokaryotic expression recombinant plasmid pET28a-CDC11 was constructed.The recombinant plasmids pET28a-CDC11 and pGEX-4T-3-CRN1 weretransformed into E.coli BL21(DE3)for heterologous expression,and obtained Cdc11 p and Crn1 p.Finally,interaction between Crn1 p and Cdc11 p was confirmed by Pull-down assay.(3)Interaction between Crn1 p and Cdc11 p were preliminarily explored by Co-IP.The recombinant plasmid pET28a-CDC11 and plasmid pYES3/CT were cut with restriction enzyme SacI and NotI.The recombinant plasmid expressed in Saccharomyces cerevisiae plasmid pYES3/CT-CDC11 was constructed and transformed into BJ5457.At the same time,the Myc tag was fused at the C end of the CRN1,and the yeast strain BJ5457 with CRN1-Myc and pYES3/CT-CDC11 was constructed.By observing the morphological changes before and after induction,it was found that when the Cdc11 p was overexpressed,the cell phenotype was abnormal and the cell buds were prolonged and most of the cells died.Co-IP did not detect the target band,and the total protein band was also less.As a result,we hypothesized that the results were due to abnormal cell phenotypes.In brief,this study confirms that the actin regulatory protein Coronin interacts with Cdc11 from the cell cycle regulating Septin family.Combined with previous study,we confirm that Asr1 p which influences cell cycle interacts with Coronin,and obtain a signaling pathway that delineate how the intact actin skeleton regulates cell cycle.