Schrock Madison N, Yan Yao, Goeckel Megan E, Basgall Erianna M, Lewis Isabel C, Leonard Katherine G, Halloran Megan, Finnigan Gregory C
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506 USA.
Present address: School of Biological Sciences, University of Utah, Salt Lake City, UT, 84112, USA.
Access Microbiol. 2022 Mar 24;4(3):000341. doi: 10.1099/acmi.0.000341. eCollection 2022.
The cytoskeleton serves a diverse set of functions in both multi- and unicellular organisms, including movement, transport, morphology, cell division and cell signalling. The septin family of cytoskeletal proteins are found within all fungi and metazoans and can generate three-dimensional scaffolds that promote membrane curvature, serve as physical barriers and coordinate cell cycle checkpoints. In budding yeast, the septins organize into polymerized filaments that decorate the division site between mother and daughter cells during mitosis; assembly of this structure at the 'bud neck' is critical for completion of cytokinesis and execution of numerous other cellular events. One such pathway includes bud site selection and the recruitment of proteins such as Bud4 and Bud3 that are responsible for promoting an axial budding pattern in haploid yeast. While Bud4 appears to be recruited to the septins independently of the presence of Bud3, it is likely that Bud3 can localize to the bud neck using both Bud4-dependent and Bud4-independent mechanisms. Furthermore, it remains unclear which precise domain or domains within Bud3 is/are both necessary and sufficient for optimal association at the septin structure. In this study, we examined the localization of GFP-Bud3 constructs in otherwise wild-type (WT) haploid yeast cells expressing Cdc10-mCherry using fluorescence microscopy; we tested a collection of N- and C-terminal truncations and fusions of separate Bud3 protein elements to identify the smallest domain(s) responsible for bud neck localization. We found that the coordinate action of the central amphipathic helix (residues 847-865) and a partially conserved C-terminal motif (residues 1172-1273) was sufficient to promote bud neck recruitment in the presence of endogenous Bud3. This domain is considerably smaller than the previously characterized C-terminal portion required to physically interact with Bud4 (1221-1636) and utilizes a similar mechanism of pairing membrane association, with a separate localization domain, similar to other non-septin proteins targeted to the division site during cell division.
细胞骨架在多细胞生物和单细胞生物中都发挥着多种功能，包括运动、运输、形态形成、细胞分裂和细胞信号传导。细胞骨架蛋白中的Septin家族存在于所有真菌和后生动物中，能够生成三维支架，促进膜弯曲，充当物理屏障并协调细胞周期检查点。在芽殖酵母中，Septin蛋白组装成聚合丝，在有丝分裂期间装饰母细胞和子细胞之间的分裂位点；这种结构在“芽颈”处的组装对于胞质分裂的完成和许多其他细胞事件的执行至关重要。其中一条途径包括芽位点选择以及招募负责促进单倍体酵母中轴向芽殖模式的蛋白质，如Bud4和Bud3。虽然Bud4似乎独立于Bud3的存在而被招募到Septin蛋白中，但Bud3可能通过依赖Bud4和不依赖Bud4的机制定位于芽颈。此外，尚不清楚Bud3中哪些精确的结构域对于在Septin结构上的最佳结合既是必要的又是充分的。在这项研究中，我们使用荧光显微镜检查了GFP-Bud3构建体在表达Cdc10-mCherry的野生型（WT）单倍体酵母细胞中的定位；我们测试了一系列N端和C端截短以及单独的Bud3蛋白元件的融合，以确定负责芽颈定位的最小结构域。我们发现，中央两亲性螺旋（第847-865位氨基酸残基）和部分保守的C端基序（第1172-1273位氨基酸残基）的协同作用足以在存在内源性Bud3的情况下促进芽颈招募。该结构域比先前确定的与Bud4物理相互作用所需的C端部分（1221-1636）小得多，并利用了类似的膜结合配对机制，具有一个单独的定位结构域，类似于细胞分裂期间靶向分裂位点的其他非Septin蛋白。
