Signal transduction mechanism underlying thiamin starvation-induced metabolic regulation in Saccharomyces cerevisiae

在酿酒酵母中，参与合成焦磷酸硫胺素和从胞外环境中利用硫胺素的基因(Thi基因)的表达是协调诱导的，以响应硫胺素饥饿，这一机制被称为酵母Thi调节系统。在该体系中，硫胺素焦磷酸(TPP)作为辅阻遏物，已鉴定出三个正调控因子(Thi2p、Thi3p和Pdc2p)。Thi3p感应细胞内信号TPP，在Thi2p和Pdc2p中存在一个推测的DNA结合域。然而，这些调控因子如何在导致Thi基因转录调控的信号转导途径中发挥作用尚不清楚。前年，我们发现Pdc2p具有转录活性并与Thi3p相互作用，这两种活性被外源硫胺素部分减弱。此外，在TH3干扰物中，Pdc2p的转录活性降低。然后，我们尝试用一系列缺失突变体来确定Pdc2p的功能结构域(926个氨基酸)。酵母单杂交和双杂交分析表明，407-581区和582-889区分别是转录活性和与Thi3p相互作用所必需的。当Thi3p相互作用区域从Pdc2p中去除后，其转录活性不受THI3基因缺陷的影响。此外，还对重组蛋白Pdc2p(407-581)和Pdc2p(407-926)的唯一色氨酸残基(Trp-450)进行了丙烯酰胺荧光猝灭研究。从Pdc2p(407-926)中去掉582-926区域后，猝灭剂的可及性增加，表明Thi3p相互作用的结构域掩盖了转录激活结构域。很可能是与Thi3p的相互作用导致Pdc2p-DNA复合体的构象改变，导致转录激活结构域暴露在溶剂中。

In Saccharomyces cerevisiae the expression of genes involved in the synthesis of thiamin pyrophosphate and the utilization of thiamin from the extracellular environment (THI genes) is coordinately induced in response to thiamin starvation, a mechanism called the yeast THI regulatory system. In this system thiamin pyrophosphate (TPP) serves as a corepressor, and three positive regulatory factors (Thi2p, Thi3p and Pdc2p) have been identified. Thi3p senses the intracellular signal, TPP, and there exists a putative DNA-binding domain in Thi2p and Pdc2p. However, it is not known how these regulatory factors function in the signal transduction pathway leading to the transcriptional regulation of THI genes.In the previous year, we found that Pdc2p has the transcriptional activity and interacts with Thi3p, and that these two activities are partially diminished by exogenous thiamin. In addition, the transcriptional activity of Pdc2p is decreased in thi3 disruptant. Then we tried to determine the functional domains of Pdc2p (926 amino acids) using a series of deletion mutants. It was suggested, by yeast one-hybrid and two-hybrid analyses, that the regions from 407 to 581 and that from 582 to 889 are necessary for the transcriptional activity and the interaction with Thi3p, respectively. When the Thi3p-interacting domain was removed from Pdc2p, the transcriptional activity was unaffected by the defect of THI3 gene. Moreover, a fluorescence quenching study with acrylamide was carried out on a sole tryptophan residue (Trp-450) of recombinant proteins, Pdc2p(407-581) and Pdc2p(407-926). The removal of the 582-926 region from Pdc2p(407-926) resulted in an increase in accessibility of the quencher, suggesting that the Thi3p-interacting domain masks the transcriptional activation domain. It is likely that the interaction with Thi3p causes a conformational change in the Pdc2p-DNA complex leading to exposure of the transcriptional activation domain to solvent.