Characterization Of The Role Of Boi2 In The Regulation Of Polarized Growth In Budding Yeast

The budding yeast Saccharomyces cerevisiae reproduces vegetatively through budding.Budding is a highly polarized growth,which depends on the polarized organization of the actin cytoskeleton,directed delivery of secretory vesicles and the fusion of secretory vesicles to the plasma membrane.The elucidation of the mechanisms by which polarized growth is regulated is scientifically significant to the understanding of the mechanisms of cell growth and poliferation.Previous studies have shown that a number of proteins including Boi1 and Boi2 are involved in the regulation of polarized growth in S.cerevisiae.Boi1 and Boi2 are a pair of paralogs that share identical domain organization and similar cellular functions.They are composed of a src-homology 3（SH3）domain,a sterile alpha motif（SAM）domain,a proline-rich（PR）domain and a pleckstrin-homology（PH）domain.Boi1 and Boi2 localize the sites of polarized growth and regulate exocytosis.However,in both proteins,particularly in Boi2,the domains that confer their polarity-site localization as well as the domains that drive their function in polarized growth are not clear.In this study,we focus on Boi2 and investigated the domains that confer Boi2’s polarity-site localization and function in polarized growth.We also analyzed the interactions between Boi2 and the Rho GTPases Cdc42 and Rho3 aiming to elucidate the mechanism by which Boi2 regulates polarized growth.We first dissected the domains within Boi2 that confer Boi2’s bud-cortex localization.A previous study has shown that the PH domain of Boi2 is able to bind the phosphoinositide PIP₂,which is rich in the plasma membraneand and plays a crucial role in the targeting of Boi2 to the cell surface.However,our results show that the PH domain alone does not localize on the plasma membrane.It seems that another domain may also be required.Interestingly,we identified a novel coiled-coil（CC）domain C-terminally adjacent to the PH domain.We found that the CC domain cooperates with the PH domain to confer bud-cortex localization.In addition,the PH-CC bi-domain can also largely support the growth of the Boi1-and Boi2-depletd cells like full-length Boi2 does.Both the PH domain and the CC domain are critical for this Boi2 function.Thus,the PH-CC bi-domain appears to be the major functional domain that confers Boi2’s bud-cortex localization and function in cell growth.How does the PH-CC bi-domain function?A previous study has shown that Boi1 interacts physically with the Rho GTPase Cdc42.This interaction is dependent on Boi1’s C-terminal region and Boi1 only binds to the GTP-bound form of Cdc42.This result suggests that Boi1and Boi2 may be the downstream target of Cdc42.By using recombinant proteins produced in E.coli cells and protein pull-down assay,we found that Boi2 can also bind to Cdc42.This interaction is mediated by the PH domain plus a short sequence C-terminally adjacent to the PH domain.In contrast to the previous report,we observed that Boi2 does not prefer to bind the GTP-bound form of Cdc42.We obtained identical result in vivo using the GST pull-down assay.Our result suggests that Boi2 might not be a downstream effector of Cdc42.Besides Cdc42,Boi2 also binds to the Rho GTPase Rho3.The PH domain of Boi2 is sufficient to mediate the binding to Rho3.The binding between Boi2 and Rho3 is also independent of the GTP-bound form of Rho3.These results suggest that Boi2 may function in polarized growth by the interaction with Cdc42 and Rho3 via the PH domain.We found that the CC domain within the PH-CC bi-domain interacts homotypically.This interaction may contributes to the dimerization or oligomerization of Boi2 in vivo.We observed that high-copy BOI2 suppressed the growth defect of Rga1-C538-overexpressing cells as well as the secretion-defective sec15-1 mutant.These two mutants can also be suppressed by high-copy RHO3 but not CDC42,suggesting that Boi2 may function in the Rho3-controlled exocytosis pathway.Interestingly,the SAM-PH-CC region of Boi2 is the functional domain that is necessary and sufficient to suppress the two mutants.Both the SAM domain and the PH-CC bi-domain are required whereas the Bem1-binding proline-rich domain is dispensable for this function.In addition,we found that Boi2 localizes to the bud neck at early cell-cycle stage and this localization happens to be mediated by the SAM-PH-CC region.This finding indicates the importance of the SAM domain in Boi2’s localization and function in polarized growth.The SAM domain of Boi2 also interacts homotypically.This interaction may play a role in the assembly of Boi2,Cdc42 and Rho3 into a large protein complex.Our results reveal the domains that confer Boi2’s polarity-site localization and the domains that drive Boi2 function in polarized growth.They also clarify the physical interaction between Boi2 and the Rho GTPases Cdc42 and Rho3.Our results highlight the importance of the PH,CC and SAM domains in Boi2’s localization and cellular function and gain new insight in the mechanisms by which Boi2 regulates polarized growth.