Girgin Sevilay Münire, Çaydaşi Ayşe Koca
Department of Molecular Biology and Genetics, Collage of Sciences, Koç University, İstanbul, Turkiye.
Turk J Biol. 2024 Aug 5;48(4):267-278. doi: 10.55730/1300-0152.2702. eCollection 2024.
BACKGROUND/AIM: Spindle pole bodies (SPB), the functional equivalent of centrosomes in yeast, duplicate through generation of a new SPB next to the old one. However, SPBs are dynamic structures that can grow and exchange, and mechanisms that regulate SPB size remain largely unknown. This study aims to elucidate the role of Bud14 in SPB size maintenance in .
We employed quantitative fluorescence microscopy to assess the relative and absolute amounts of SPB structural proteins at SPBs of wildtype cells and in cells lacking (). Quantifications were performed using asynchronous cell cultures, as well as cultures synchronously progressing through the cell cycle and upon different cell cycle arrests. We also utilized mutants that allow the separation of Bud14 functions.
Our results indicate that higher levels of SPB inner, outer, and central plaque proteins are present at the SPBs of cells compared to wildtype cells during anaphase, as well as during nocodazole-induced M-phase arrest. However, during α-factor mediated G1 arrest, inner and outer plaque proteins responded differently to the absence of . A Bud14 mutant that cannot interact with the Protein Phosphatase 1 (Glc7) phenocopied in terms of SPB-bound levels of the inner plaque protein Spc110, whereas disruption of Bud14-Kel1-Kel2 complex did not alter Spc110 levels at SPBs. In cells synchronously released from α-factor arrest, lack of Bud14-Glc7 caused increase of Spc110 at the SPBs at early stages of the cell cycle.
We identified Bud14 as a critical protein for SPB size maintenance. The interaction of Bud14 with Glc7, but not with the Kelch proteins, is indispensable for restricting levels of Spc110 incorporated into the SPBs.
背景/目的：纺锤极体（SPB）是酵母中与中心体功能相当的结构，通过在旧的SPB旁边生成一个新的SPB进行复制。然而，SPB是动态结构，能够生长和交换，而调节SPB大小的机制在很大程度上仍不清楚。本研究旨在阐明Bud14在酿酒酵母中维持SPB大小的作用。
我们采用定量荧光显微镜来评估野生型细胞以及缺乏Bud14的细胞的SPB处SPB结构蛋白的相对和绝对含量。使用异步细胞培养物以及同步经历细胞周期和处于不同细胞周期停滞状态的培养物进行定量分析。我们还利用了能够分离Bud14功能的突变体。
我们的结果表明，与野生型细胞相比，在后期以及诺考达唑诱导的M期停滞期间，Bud14缺失细胞的SPB处存在更高水平的SPB内部、外部和中央板层蛋白。然而，在α因子介导的G1期停滞期间，内部和外部板层蛋白对Bud14缺失的反应不同。一个不能与蛋白磷酸酶1（Glc7）相互作用的Bud14突变体在内部板层蛋白Spc110的SPB结合水平方面模拟了Bud14缺失的情况，而Bud14-Kel1-Kel2复合物的破坏并未改变SPB处Spc110的水平。在从α因子停滞中同步释放的细胞中，缺乏Bud14-Glc7会导致细胞周期早期SPB处Spc110增加。
我们确定Bud14是维持SPB大小的关键蛋白。Bud14与Glc7而非与Kelch蛋白的相互作用对于限制掺入SPB的Spc110水平是必不可少的。
