| Polarized cell growth leads to local expansion of cell surface, such as filopodia and lamellipodia in animal cells. As to the budding yeast Saccharomyces cerevisiae, similar process leads to the construction of a bud and its separation from the mother. S.cerevisiae budding process is a perfect paradigm for the mechanisms of polarity growth correlated with cell-cycle progression. The size and shape of bud correlated with the cell-cycle progression. During each budding cycle, cell polarity establishes, relieves, rebuilds and exits as cell-cycle progressing, leading to bud emergence, enlarging and separation from mother cell. This process requires a perfect co-ordination among cytoskeleton organization, secretion and endocytosis. Rho GTPases are key regulators in this progression. S.cerevisiae has six Rho GTPases, named Cdc42and Rho1-Rho5. Among them, only Cdc42and Rho1are essential. Cdc42was considered to be the master regulator in polarity establishment. Rho1plays a main role in regulating the synthesis and integrity of cell wall, and Rho2has overlapping function with Rho1. Rho5shares high identity with Cdc42in amino sequence and may play roles in response to osmotic or peroxide stress. Rho3and Rho4share essential role in maintenance cell polarity and actin cytoskeleton organization.Rho4localizes throughout the cell membrane and enriches to polarized growth site:bud tip during bud emergence, bud cortex during isotropic growth of bud, and bud neck at bud separation from mother cell. Rho4is thought to play a role in maintenance of cell polarity, according to the death with small bud phenotype of rho3A rho4A cells. But the localization of Rho4indicated us that Rho4may also play roles in other process during budding progression besides maintenance of polarized growth. Unfortunately, we have no morphology proofs in the budding yeast to support the hypothesis. Rho4’s role in actin organization appears to be conserved in other yeasts and in the filamentous fungi. For example, in the fission yeast Schizosaccharomyces pomhe, deletion of SpRHO4resulted in disappear of actin at the growing cell ends. Rho4homologs in those species also play role in cell separation. In S. pombe, deletion of SpRHO4resulted cell separation defect at elevated temperature; in pathogenic yeast Candida albicans, deletion of CRL1/CaRHO4resulted in cell chain defect. In the filamentous fungi Neurospora crassa, NcRho4play an essential role in cytokinesis as complete abolishment of actin ring formation and septum construction in its null mutant.Rho4is unique among the six Rho GTPases in the budding yeast for its longest N-terminal extension, which is73a.a. long and69a.a. longer than Cdc42’s and Rho5’s. It’s common that Rho4homologs in other yeast and filamentous fungi share long N-terminal extension. But we have no idea of the role of long N-terminal extension. Rho4was found to be regulated by Rdi1, the sole Rho GDI in the budding yeast. Rdi1overexpression caused the decrease of Rho4protein level. Rdi1and Ygk3(a GSK-3p kinase) promoted he degradation of Rho4through proteolysis. So far, the only Rho GAP for Rho4, and Rho3as well, is Rgdl. No GEFs and other Rho GAPs for Rho4have been identified.In this study, we try to reveal the Rho4’s function and its regulation mechanism. We assess the function of Rho4through overexpression active or inactive rho4mutants and isolation of rho4-Ts mutants. We identify the function of Rho4’s N-terminal extension. Meanwhile, yeast two-hybrid and multicopy suppression screen were carried out to identify the Rho4interacting proteins. In this study, we found that overexpression of constitutively active rho4Q131L mutant in rdil A cells leading to growth defect and large, round and no bud defect, indicating excess of Rho4activity could block bud emergence. Most of the rho4-Ts mutants we isolated display large and round morphology, and some mutants display low percentage of multi-budded cells in cell population, indicating that Rho4may play other roles in addition to maintenance of polarized growth. Compared the defects of rho3A rho4Q131L, rho3A rho4-m6and rho3A rho4-m8cells, we found that site mutation in N-terminal extension also contributed to cell defects of rho3A rho4-m6and rho3A rho4-m8cells in addition to Q131R mutation. We show that long N-terminal extension plays an important role in Rho4function since deletion of the first61a.a. of N-terminal extension did not affect on the localization of Rho4and rho3Δ rho4-Δ61cells expressing truncated Rho4-Δ61exhibited morphological defect at24℃and a growth defect at37℃. We also found that rho3A rho4-Δ42cells expressing truncated Rho4-Δ42lacking the region1-42a.a., which containing a potential PEST motif from9a.a. to40a.a, exhibited growth and morphology as control rho3A RH04cells. Our results indicated that the key region for N-terminal extension function is form43a.a. to73a.a.. To identify new interacting proteins of Rho4, we performed yeast two hybrid screen, leading to isolation of a Rho GAP Bem2. Bem2specially interacts with the GTP-bound form of Rho4and the interaction is mediated by its Rho GAP domain. Overexpression of BEM’2full length or BEM2-C2, containing only Rho GAP domain, aggravates the defects of rho3A rho4mutants. These results suggest that Bem2might be a novel Rho GAP for Rho4. Bem2also interacts with GTP-Rho2in our test, indicating that Bem2might also a Rho GAP of Rho2. |
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