Localization And Function Of Bem3,a Rho GTPase-activating Protein In Budding Yeast

Saccharomyces cerevisiae reproduces through budding.The budding process involves the establishment of cell polarity and polarized growth.Cdc42,the central molecule regulating cell polarity in budding yeast,is involved in the regulation of actin cytoskeleton,septin organization and polarized secretion.Bem3 is a GTPase-activating protein(GAP)for Cdc42.It plays critical roles in the spatial and temporal control of Cdc42.Previous studies showed that Bem3 localizes to the sites of polarized growth including the presumptive bud site,the bud tip in small-budded cells and the bud neck in large-budded cells during cytokinesis,Bem3 also plays a role in septin organization.However,how Bem3 localizes to the polarity sites and how Bem3 regulates septin organization are not clear.Therefore,we performed a structure-function analysis on Bem3,aiming to reveal the mechanisms underlying Bem3's localization and its regulation on septin organization.First,we investigated the targeting domain of Bem3.We generated a series of GFP-Bem3 segment fusions that contain the N-terminal region and the central region of Bem3 and examined if they can localize to the polarity sites in wild-type cells by fluorescence microscopy.Two distinct targeting domains were identified.The N-terminal coiled coil-containing targeting domain 2(TD2)is the major targeting determinant of Bem3.The central PX-PH domain-containing targeting domain 1(TD1)is an auxillary targeting determinant.PX and PH domains were shown to have affinity for phosphatidylinositol that is rich in the plasma membrane by previous studies.Thus,the targeting ability of the PX-PH-containing TD1 is well expected.However,the better targeting capacity of the coiled-coil-containing TD2 is surprising to us.We speculated that some proteins may interact with the coiled-coil domain and recruit TD2 to the polarity sites.In order to explore this possibility,we performed a yeast two-hybrid screen using TD2-containing Bem3-N6 segment as bait.From this screen,we identified Epol as a Bem3 interactor.Epol and full-length Bem3 interact with each other at the polarity sites in vivo as shown by the bimolecular fluorescence complementation(BiFC)assay.Epol contains four coiled-coil domains.It is a member of the protein complex,polarisome,which plays a role in the anchoring of cortical endoplasmic reticulum at the bud tip.We found that Epol interacts with the coiled-coil domain of Bem3 via its third and fourth coiled-coil domains.When EPO1 was deleted,the fluorescence intensity of full-length Bem3 at polarity sites was reduced significantly.The polarity-site localization of TD2 was completely abolished.Therefore,we speculate that Bem3 was recruited to the sites of polarized growth via the interaction with Epol.In the course of our study with the TD2 domain,we found that the coiled-coil domain of Bem3 interacts homotypically,implying that Bem3 may oligomerize in,vivo.Overexpression of full-length Bem3 blocked cell growth.Cells lost polarity and became large,round and unbudded,presumably due to the inactivation of Cdc42.When we overexpressed the Bem3?CC segment,which lacks the coiled-coil domain,we found that the growth-blocking function of Bem3 was dramatically reduced.Cells displayed a partial loss of cell polarity.The removal of the coiled-coil domain would eliminate Bem3's interaction with the polarisome member,Epol,and block Bem3's oligomerization.We observed that overexpression of full-length Bem3 had a stronger effect in growth inhibition than overexpression of Bem3?CC in epo1 ?.cells.This finding suggests that Bem3 oligomerization via homotypic coiled-coil interaction is important for Bem3's regulation on Cdc42.After Bem3's TD1 and TD2 had been identified,we examined their roles in the regulation of septin organization.By complementation testing of a series of BEM3 segments in bem3? rga1? gin4? mutant driven by BEM3's own promoter,we found that the Bem3ATD1 and Bem3?N6 segments,complemented the elongated-bud defect much less efficiently compared with full-length Bem3.The Bem3-N segment lacking the C-terminal RhoGAP domain can not complement the defect.This result suggests that the functions of TD1 and TD2 depend on the RhoGAP domain.Moreover,we found that overexpression of Bem3-N segment in wild-type cells caused elongated-bud defect.The septins in cells with elongated buds were disorganized.Bem3-N sometimes co-localized with the septins,indicating that Bem3-N functionally interacts with the septins.Thus,an excess of Bem3-N in the cells is sufficient to disrupt normal septin-ring assembly without engaging the RhoGAP domain.Furthermore,we found that the effect of Bem3-N overproduction on septin organization was mainly mediated by the TD2-containing Bem3-N6 but not by TD1 since overexpression of Bem3-N6 caused similar bud elongation defect whereas overexpression of Bem3TD1 did not cause such a defect.This result suggests that the TD2 of Bem3 is implicated in the regulation of septin organization in a manner without engaging the RhoGAP activity of Bem3.How does TD2 regulate septin organization?The polarisome component,Epol,interacts with Bem3.In the course of this study,a research group reported that Pea2,another polarisome component,also interacts with Bem3.Since an important function of the polarisome is to regulate septin organization,the interaction between Bem3 and two polarisome components suggests that the polarisome may be involved in Bem3's function in septin organization.We found that the proportions of cells with elongated buds in epo1? and pea2? mutants overexpressing Bem3-N6 were significantly reduced.This result suggests that an excess of Bem3-N6 may affect septin-ring assembly indirectly through disturbing the functions of the polarisome.Since the deletion of EPO1 or PEA2 did not eliminate the elongated-bud defect in cells overexpressing Bem3-N6,we speculate that the TD2 domain may be able to interact with other proteins that regulate septin-ring assembly.From the yeast two-hybrid screen using Bem3-N6 as bait,Kssl,a mitogen-activated protein kinase(MAPK)implicated in pheromone response and filamentous growth,was also identified.Before our study,Kssl was thought to localize to the nucleus.However,we found that,in addition to an enrichment in the nucleus,Kssl also localized to the polarity sites,including the bud tip in small-budded cells and the bud neck in large-budded cells around cytokinesis.BiFC assay confirmed that Bem3 interacts with Kssl in the cells and the interaction occurs at the septin-ring assembly site.Since neither the elongated bud defect caused by Bem3-N6 overexpression nor the localization of Bem3-N6 was affected by KSS1 deletion,the physiological role of Bem3-Kssl interaction remains elusive at this time.Lastly,we found that Bem3 can interact with the active GTP-bound forms of Rho3 and Rhol via its RhoGAP domain,indicating that Bem3 may also act as a GAP for Rho3 and Rhol besides Cdc42.Our studies on the genetic interaction of BEM3 with RHO1 and RHO3 suggest that Bem3 may not be involved in Rhol-directed cell wall synthesis,but may specifically down-regulate Rhol-directed actin cytoskeleton organization.The cellular process that requires Bem3-Rho3 interaction is not identified in this study.Further investigation is needed to address this issue.