Characterization Of The Mechanism That Msb1Controls Polarized Growth In The Budding Yeast Saccharomyces Cerevisae

Polarized cell growth is a common and important process for many cell types, the temporal and spatial regulation of which is necessary for normal cell growth. In the budding yeast Saccharomyces cerevisae, polarized growth is manifested by the assembly and growth of a bud, a process controlled by complex signal pathway and small Rho GTPases. As the members of this family, Cdc42and Rhol are two key Rho GTPases implicated in the regulation of polarized growth. Cdc42is thought to be the central regulator in cell polarization and it directs bud emergence and growth via regulating the assembly of cytoskeleton including septin and actin. Rhol is thought to involve in cell wall generation and integrity. Responding to extracellular and intracellular signal, Rhol transfers signals to downstream targets Pkcl to regulate cell integrity or Fksl to direct1,3-β-glucan synthesis. Many regulators and effectors of Cdc42and Rhol have been found, but there are still lots of unknown factors and mechanisms for us to identify and explain.Msbl is a protein that contains a RhoGAP domain at its N-terminus. It is dispensable for cell growth. Although the deletion of MSB1did not produce defects in cell growth or morphology, high-copy of MSB1could suppress the growth defect of several cdc42-Ts and cdc24-Ts mutants including cdc42-1, cdc42-201, and cdc42G60D. msblΔ also showed synthetic lethal interaction with cdc24, beml, and bem2mutations. Thus, it appears that Msbl positively regulates Cdc42function. To explore how Msbl regulates Cdc42, we first examined the subcellular localization of Msbl and found it localizes at the sites of polarized growth similar to Cdc42. By using GST pull down assay, we found Msbl can form complex with Cdc42in vivo. Although containing a conserved Rho GAP domain at its N terminus, we proved that GAP activity is not needed in the function of Msbl on Cdc42through mutation on the conserved amino acid in Msbl required for the putative RhoGAP activity. Because msblΔ bem1Δ cells are dead, Msbl and Beml’s functions are redundant. Beml functions as scaffold to bring Cdc42, its regulators, and effectors together to promote Cdc42function. We speculate Msbl has similar function so we employ GST pull down assay to check interaction between Msbl and Beml, Cdc24, Boil/Boi2. We found Msbl can form complex with Boi1/Boi2. And we also identified their binding site which is Boil/Boi2’s functional region and this region contains PH domain which interacts with Cdc42but excludes proline rich domain which binds to Beml, indicating Msbl and Beml can bind to Boil/Boi2at the same time. And then through genetics experiments, we found Boi2can promote Msbl’ function and impact the ability of Msbl suppressing cdc42-201mutant. Based on the data above, we propose that Msbl promotes Cdc42function via interacting with Boi1/Boi2.In addition to the regulation on Cdc42function, we also want to know whether Msbl is implicated in other cellular functions. Since the deletion of MSB1did not produce an obvious phenotype, we studied the effect of MSB1overexpression on cell growth and morphology. We found that cell overexpressing MSB1did not display a defect in cell growth. However, overexpressing MSB1induced bud elongation and the formation of short cell chain. Through observing the localization of septin, we found that the septins were misorganized in the elongated buds, indicating Msbl may takes part in septin organization. The treatment of cells with lylicase digestion suggests that the formation of cell chain is due to a defect in cell separation. We also found abnormal deposition of chitin and glucan in cells overexpressing Msbl, indicating Msbl may also regulate these cell wall components syntheses. To know more about Msbl, we characterize the localization and function of different Msbl fragments. We found the N terminus including Rho GAP domain only localize at bud neck and C terminus localize as full length except it cannot localize at bud cortex and neck at the same time. Although full length of MSB1is necessary for it suppress cdc42-201, different localizations lead to fragments with different functions. Through overespression we found that N terminus can lead to bud elongation although septin does not be impacted and C terminus may be the reason of cell-chain and abnormal deposition of cell wall caused by Msbl.Msbl not only regulates Cdc42function, it could also regulate Rhol function. We found that Msbl forms a complex with Rhol in vivo by GST pull-down assay. In addition, overexpressing Msbl inhibited the growth of rhol-3and rhol-104mutant. In these mutants overexpressing Msbl, the percentage of cells with a small bud increased and β(1,3)-glucan synthesis in buds decreased, suggesting that Msbl overexpression inhibited Rhol function in the synthesis of glucan in the cell wall. Considering that high-copy Msbl promotes Cdc42function but represses Rhol function, we propose that Msbl may play a role in the coordination of Cdc42and Rho1functions during polarized growth at the small-budded stage to ensure proper bud growth.We employed yeast two-hybrid screen to identify proteins that could interact with Msb1full-length, Msb1-N, or Msb1-C fragments, but we have not found any interesting candidates. Through EMS mutagenesis, we have got a msb1Δ cdc42E62mutant which cannot growth at30℃. We also screened mutants that became inviable upon MSB1overexpression. This work is still in progress.We also tried to isolate new genes that could rescue the growth defect of temperature-sensitive cdc42K186R mutant on high-copy plasmids. The Cdc42K186R mutant protein displayed faster GTP hydrolysis rate than wild-type Cdc42. From this screen, we isolated SKG6, MID2, and MLF3which can rescue the mutant. SKG6and MLF3are reported to interact genetically with GIC1and GIG2, two genes whose encoded proteins bind Cdc42. Mid2is thought to regulate Rho1function in cell wall integrity.