Snf1 複合體在酵母菌生長表現型及假菌絲生成上之功能性鑑定

Abstract

Snf1激酶是一個絲胺酸/酥胺酸蛋白質激酶家族的一員。在許多高等真核生物中， Snf1 是高度被保留的。AMP-activated protein kinase (AMPK)AMPK 是酵母菌 Snf1在哺乳動物細胞中的同源蛋白。在酵母菌中，當環境中葡萄糖含量很低或是只存在其它醣類的碳來源時，Snf1 的激酶活性會被活化。當環境中有葡萄糖存在時，Snf1 的激酶活性是被抑制的。在一些寄生性的真菌中，假菌絲生成是一種很重要的致病行為，並且當整個 Snf1 基因被刪除時，假菌絲生成會出現嚴重的缺陷。為了要研究 Snf1 激酶活性和酵母菌假菌絲生成上的關係，我利用了三種已知的激酶突變種 (G53R、K84R 和 T210A) 來觀察激酶活性在假菌絲生成上有何影響，同時也觀察 V222I 和 P251L 這兩個突變種的影響。結果顯示，Snf1激酶活性是被假菌絲生成所需要的。具有功能的 Snf1 激酶必須是一個完整的蛋白複合體，由三個不相同的蛋白所組成，其中包含了 Snf1 激酶本身、活化蛋白 Snf4 和其中一種調節蛋白 Sip1、Sip2 和 Gal83。Gal83 是其中唯一一種被認為可以進入細胞核的調節蛋白。Gal83 基因的刪除會造成細胞生長缺陷。為了解 Snf1 複合體在反應環境改變時，在調控基因方面是否和 Gal83 進入細胞核的現象有關係，我利用間接免疫螢光染色的方法，藉由將 Gal83 的基因刪除掉後和 Gal83 野生型比較，來分析 Snf1 在酵母菌細胞中的分布，之後並放入帶有標定的 Gal83 來做共同螢光染色。結果顯示，Gal83 可能扮演一個束縛蛋白，在葡萄糖存在時，Gal83 將 Snf1 束縛在細胞質中，當葡萄糖不存在或是只存在其它醣類的碳來源時，Gal83 進入細胞核的同時，也將和其結合並具有完整功能的 Snf1 一同帶入。並且結果也暗示著 Snf1 在細胞質和細胞核中扮演著不同的功能。

Snf1 kinase is a member of serine/threonine protein kinase family. In many advanced eukaryotic organisms, Snf1 is highly conserved. AMP-activated protein kinase (AMPK) is the homolog of yeast Snf1 in mammalian cells. In yeast, Snf1 is a glucose-repressible kinase, and it is activated by the glucose starvation or the alternative carbon sources. In some parasitic fungi, the pseudohyphal formation plays an important role in pathogenetic behavior for their host. To investigate the relationship between the Snf1 kinase activity and pseudohyphal formation, I used three well-known kinase activity mutants (G53R, K84R, and T210A)) to study their effects on pseudohyphal formation. V222I and P251L were also studied. These results said that the Snf1 kinase activity was required for the pseudohyphal formation. The functional Snf1 kinase is also known as a protein complex made up by three different subunits, the Snf1 itself (catalytic alpha subunit), the Snf4 (regulatory gamma subunit), and one of the Sip1, Sip2, or Gal83 (targeting beta subunit). The Gal83 is the only one subunit that can target to nucleus, and the Gal83 deletion causes the growth defects in low glucose condition. To understand the regulatory mechanism of the Snf1 protein complex in responsive of the environmental stresses, I use the indirect immunofluorescence to identify whether the function of the Snf1 kinase is associated with the Gal83 nuclear localization. However, I found that Gal83 may be an anchoring protein catching Snf1 in the cytosol in glucose-repressible condition, and the Gal83 localized to nucleus with the functional Gal83-bound Snf1 in response to the glucose starvation or the alternative carbon sources. Moreover, these results also suggest that Snf1 may play different roles in the cytosol and in the nucleus.