Institut für Mikrobiologie und Genetik, Universität Göttingen, Göttingen, Germany.
Mol Microbiol. 2010 Apr;76(1):220-35. doi: 10.1111/j.1365-2958.2010.07093.x. Epub 2010 Feb 28.
Rho GTPases have multiple, yet poorly defined functions during cytokinesis. By screening a Neurospora crassa knock-out collection for Rho guanine nucleotide exchange factor (GEF) mutants that phenocopy rho-4 defects (i.e. lack of septa, slow growth, abnormal branching and cytoplasmic leakage), we identified two strains defective in homologues of Bud3p and Rgf3 of budding and fission yeast respectively. The function of these proteins as RHO4-specific GEFs was determined by in vitro assays. In vivo microscopy suggested that the two GEFs and their target GTPase act as two independent modules during the selection of the septation site and the actual septation process. Furthermore, we determined that the N. crassa homologue of the anillinrelated protein BUD4 is required for septum initiation and that its deficiency leads to typical rho4 defects. Localization of BUD4 as a cortical ring prior to septation initiation was independent of functional BUD3 or RGF3. These data position BUD4 upstream of both RHO4 functions in the septation process and make BUD4 a prime candidate for a cortical marker protein involved in the selection of future septation sites. The persistence of both BUD proteins and of RHO4 at the septal pore suggests additional functions of these proteins at mature septa.
Rho GTPases 在胞质分裂过程中具有多种但定义不明确的功能。通过筛选粗糙脉孢菌敲除文库中与 Rho 鸟嘌呤核苷酸交换因子（GEF）突变体，这些突变体模拟了 rho-4 缺陷（即缺乏隔膜、生长缓慢、分支异常和细胞质渗漏），我们鉴定出两个分别在芽殖酵母和裂殖酵母的 Bud3p 和 Rgf3 同源物缺陷的菌株。这些蛋白质作为 RHO4 特异性 GEF 的功能通过体外测定来确定。体内显微镜观察表明，这两个 GEF 及其靶 GTPase 在隔膜位点的选择和实际隔膜形成过程中作为两个独立的模块起作用。此外，我们确定了粗糙脉孢菌的肌动蛋白相关蛋白 BUD4 的同源物对于隔膜起始是必需的，并且其缺乏导致典型的 rho4 缺陷。在隔膜起始之前 BUD4 作为皮质环的定位独立于功能性 BUD3 或 RGF3。这些数据将 BUD4 定位在隔膜形成过程中 RHO4 功能的上游，使 BUD4 成为参与未来隔膜形成位点选择的皮质标记蛋白的主要候选物。BUD 蛋白和 RHO4 在隔膜孔处的持续存在表明这些蛋白质在成熟隔膜中有额外的功能。
