過度活化的Tem1 GTPase對酵母菌孢子生成的影響

Abstract

當環境缺乏養分時，會使二倍體出芽酵母菌細胞進行減數分裂並產生孢子。對於出芽酵母菌來說這是一個營養缺乏的逆境反應，因此會誘導出一些逆境蛋白，包括Hsp26蛋白。我們實驗室先前已經發現一個與Hsp26相關的紡錘絲檢控點，其會監控第二次減數分裂過程中的紡錘絲是否適合形成孢子。之前研究也發現Tem1參與在這個檢控點中。我們想了解Tem1是如何參與在此Hsp26相關紡錘絲檢控點。因為Tem1是GTP水解酶會受GAP複合物加速GTP進行水解，我們使用兩種方法將Tem1維持在GTP狀態並探討其效應。第一，是將GAP相關基因BFA1剔除以阻止GTP水解。第二，建構TEM1-Q79L突變株，組成持續與GTP結合的Tem1。我們發現若維持Tem1-GTP狀態，會使檢控點失去功能。透過pCLB2-TEM1突變細胞關閉減數分裂中Tem1表現也會使此一檢控點失效，據此我們提出兩種可能的模型來解釋此現象，一、Tem1-GDP可以主動阻擋的方式干擾孢子形成；二、Tem1必須存在才能活化Hsp26紡錘絲檢控點，接著此檢控點會反過來調控Tem1在孢子形成中的功能。

In the budding yeast Saccharomyces cerevisiae, absence of nitrogen and presence of nonfermentable carbon source lead diploid cells to enter meiosis and spore formation, which called sporulation. It is a starvation-stress response to the yeast. During sporulation, several stress proteins are induced, including Hsp26. Our laboratory has discovered that there is a Hsp26-dependent spindle checkpoint to monitor spindle assembly at meiosis II and regulates spore formation. Tem1, a component of the mitotic spindle checkpoint, is also involved in this checkpoint. We would like to know how Tem1 is involved in this Hsp26-dependent spindle checkpoint. Because Tem1 is a GTPase, which switches between GTP and GDP status, we adopted two approaches to keep Tem1 in the GTP-bound form. One was to delete the GAP-related gene, BFA1, and to block GTP hydrolysis. The other one was to construct TEM1-Q79L mutant that keeps Tem1 in the GTP-bound form. We found that Tem1-GTP may inactivates this checkpoint. pCLB2-TEM1 mutants, which shut down Tem1 during meiosis, also inactivate this checkpoint. We propose two models to explain these results. First, Tem1-GDP may actively block spore formation. Alternatively, Tem1 is required to activate the Hsp26-dependent spindle checkpoint that in turn regulates Tem1 function in spore formation.