Characterization Of The Interaction Between Bud3p And Septin In The Budding Yeast Saccharomyces Cerevisae

The septins are a group of important cytoskeleton proteins, which were originally identified in the budding yeast Saccharomyces cerevisiae. Recent studies have suggested that septins are involved in diverse cellural processes, mainly by acting as a scaffold and/or diffusion barrier. However, the regulatory mechanism of septin assembly and disassembly remains elusive today. Bud3p is recuited to bud neck by septin cytoskeleton and assembles with Ax11p, Ax12p and Bud4p into the axial landmark complex, which is involved in axial bud selection. Up to now, little has been done to explore the recruitment and assembly mechanisms. On the other hand, overexpression of Bud3p exerted deleterious effects on cell morphology and septin higher-order structure organization, as cells showed thick and elongated buds and connect into cell chains due to cytokinesis defect. Moreover, septins misorgnized into patch, spiral, bar or arc and mislocalzed to bud tip or bud cortex. This finding implied that Bud3p may play a pivotal role in septin regulation. In order to further explore the role of Bud3p in axial landmark complex assembly and genetical interaction with septin, we constructed Bud3p truncated fragments to characterize the functional domains in Bud3p required for bud neck localization, septin regulation and axial budding determination. Meanwhile, genetic and biochemical approaches were employed to characterize the interactions between Bud3p and septin. Moreover, yeast two-hybrid and multicopy suppression screen were carried out to identify the Bud3p interacting proteins.In this work, we have mapped three short and independent fragments of Bud3p including Bud3p-N/M6 (1-858 a.a), Bud3p-M19 (850-1220 a.a.) and Bud3p-C2 (1221-1466 a.a.) that are located at bud neck. The fragment Bud3p-M27 (850-1103 a.a.) in the middle portion of Bud3p is the shortest fragment identified so far, with a significant role in septin regulation. Similar to the full length Bud3p, overexpression of Bud3p-M27 also caused elongated buds and aberrant septin organization. In addition, the functional domain for axial budding is located in Bud3p-N/M6. The N-terminal of Bud3p-N/M6 contains a Dbl-homology (DH) domain and a DUF3507 (Domain of Unknown Function). These two domains are conserved in Bud3p orthologs from yeasts and filamentous fungi but function differently on axial budding. DH domain is dispensable whereas DUF3507 is necessary for axial budding. The 850-858 a.a. region shared by Bud3p-N/M6 and Bud3p-M19 is predicted to encode an amphipathic helix, which is crucial for membrane targeting, bud neck localization, septin regulation and axial budding selection.Further studies then revealed that Bud3p interacts with septin intimately. First of all, Bud3p colocalizes with the ectopic septin spirals and rings upon overexpression. Meanwhile, the roles of SHS1, CDC10 and CDC12 in septin regulation were tested when overexpressing Bud3p. The result showed that in contrast with shs1△. and cdcl2-6 cells which behaved like the wide type control, the cdc10△cells showed a partial decrease in bud elongation and a complete loss of ectopic septin spirals. GST pull-down assay suggested that Bud3p can form complex with two septin subunits, Cdc10p and Cdc11p in vivo. Bimolecular Fluorescence Complementation (BiFC) result indicated that Bud3p interacted with Cdc10p in vivo. Then, the role of Swelp-dependent checkpoint pathway was tested in BUD3-induced filamentation. Deletion of SWE1 in Bud3p-overexpressed cells caused a moderate reduction in bud elongation, but the ectopic septin spirals still existed. However, when overexpressing Bud3p-M or Bud3p-C in swelA cells, bud elongation was not observed as in wild type control, though cells overexpressing Bud3p-M still displayed ectopic septin rings and septin was located at the neck in cells overexpressing Bud3p-C.In order to understand the detailed mechanism of interactions between Bud3p and septin, we utilized yeast two-hybrid to identify the Bud3p interacting proteins. A 5' truncated BAT2 fragment which encoded Bat2p△1-212 was identified by using BUD3-M3/C2 as bait, and another 5'truncated NISI gene which encoded Nislp△1-52 was identified by using BUD3-N/M2 as bait. It was reported that Nislp located at the bud neck and has yeast two-hybrid interaction with all the five mitotic septin subunits. In this study, it was found that the neck targeting of Nislp was independent of Bud3p, Bud4p and Shslp, and that of Bud3p and its functional fragments were also irrelevant to Nislp. Moreover, the high-copy plasmid of NISI did not affect the locating pattern of Bud3p-FL, Bud3p-M and Bud3p-C, either.Meanwhile, overexpression Bud3p in shs1△cells caused temperature-dependent growth defect, as cells were inviable in 37℃. We perform a multicopy suppression screen to identify the suppressor, unfortunately, functional genes involved in this process have not been found yet. The roles of some genes related to septin regulation in inducing the bud elongation upon overexpression of Bud3p were also tested, such as CLA4, GIN4, ELM1 and KCC4 as well as septin subunit gene CDC 12, the GAP RGA1 and BEM3 of Cdc42p GTPase, and the axial budding landmark gene, BUD4, AXL1 and AXL2, However, we did not detected the effect of all the tested genes no this process.