Screening For Proteins And Genes That Physically Or Genetically Interact With Boi2,the Key Regulator Of Polarized Growth In The Yeast Saccharomyces Cerevisiae

The budding yeast Saccharomyces cerevisiae reproduces via budding,a form of highly polarized growth that involves the processes including the polarized organization of the cytoskeleton,secretory vesicle transport and fusion,and cell wall remodelling.Multiple proteins including the pair of homologous proteins Boi1 and Boi2 are involved in the regulation of polarized growth.Boi1 and Boi2 are structurally and functionally similar to each other.They contain the src-homology 3 domain,sterile alpha motif,proline-rich domain,pleckstrin-homology domain,and the coiled-coil domain.They play critical roles in polarized growth via the regulation of actin organization and secretion.However,except for several proteins including Cdc42,Bem1,and Sec1,few proteins are known to physically interact with Boi1 and Boi2.This has significantly limited the understanding of the mechanisms by which Boi1 and Boi2 regulate polarized growth.In this study,we focus on Boi2.We want to screen for proteins that physically interact with Boi2 using yeast two-hybrid screens.In addition,we want to isolate genes that genetically interact with Boi2.Our goal is to gain new insight in the understanding of the mechanisms by which Boi2 regulates polarized growth.First,to look for proteins that physically interact with Boi2,we screened a yeast two-hybrid c DNA library using Boi2 as bait.We cloned full-length Boi2 and three N-terminally truncated Boi2 segments including Boi2-S12(contains the SAM,PR,and PH-CC domains),Boi2-S7(contains the PH-CC domain),and Boi2-S24(contains just the CC domain)into the two-hybrid bait vector.We then used these baits to screen the c DNA library.We isolated a total of twelve Boi2-interacting proteins including Bem1,Bmh1,Nba1,Nis1,Dse1,Not5,Myo1,Mig1,Coq8,Gcn4,Ahk1,and Tal1.Except Bem1,there were no reports that showed the other eleven proteins interact with Boi2.We have examined the domains within Boi2 that ineract with these proteins.We found that,Bmh1,the prey that has been isolated for multiple times,intearcts with the region in Boi2 that contains the SAM,PR and the adjacent region next to PR.Nba1,Nis1,Dse1,and Not5 intearct with the SH3 domain of Boi2.Myo1 interacts with the PH domain of Boi2.Mig1 and Coq8 intearct with the PH-CC bi-domain of Boi2.Gcn4,Ahk1,and Tal1 intearct with the CC domain of Boi2.We then examined whether Boi2 may functionally interacts with any of the eleven proteins.We found that the high-copy expression of Bmh1 can reduce the toxic effect of the overexpression of full-length Boi2 and several truncated Boi2 segments on growth,suggesting that Bmh1,the 14-3-3 family member,may regulate the activity of Boi2 in the cells.Nba1,Nis1,and Dse1 interact with the SH3 domain of Boi2 and the three proteins and Boi2-SH3 all localize to the bud neck at the late cell cycle stage.However,we found that the bud neck localization of GFP-Nba1,GFP-Nis1,and GFP-Dse1 still localized to the bud neck in the boi1?boi2?mutant strain,suggesting that their bud-neck localization does not depend on Boi2.At the moment,we do not know whether Nba1,Nis1,or Dse1 may functionally interact with Boi2.Second,to look for genes that genetically interact with Boi2,we screened a yeast genomic library for high-copy suppressors that can rescue the growth defect of the boi1?boi2?mutant strain.Unfortunately,we did not succeed in this approach by using the two yeast strains boi1?boi2?and P_GAL-BOI1 boi2?in different genetic background.In spite of these failures,we did a direct test to look for high-copy suppressors that can rescue the growth defect of the P_GAL-BOI1 boi2?mutant strain among twenty-one selected genes,which are known to regulate actin organization and secretion.We found that ten genes including CDC42,BEM1,CDC24,SEC4,SEC1,SEC9,SRO7,SEC8,SSO2,and SSO1 can rescue the growth defect of the mutant strain to a varying extent.This finding suggests that Boi2 not only functionally interacts with Cdc42and its interacting partners Bem1 and Cdc24,Boi2 also functionally interacts with proteins that regulate secretion including Sec4,Sec1,and Sec9.