Randez-Gil F, Bojunga N, Proft M, Entian K D
Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität Frankfurt, Biozentrum, Niederursel, Frankfurt am Main, Germany.
Mol Cell Biol. 1997 May;17(5):2502-10. doi: 10.1128/MCB.17.5.2502.
The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.
Cat8p锌簇蛋白对于酿酒酵母利用非发酵性碳源生长至关重要。CAT8基因的表达受Mig1p介导的葡萄糖抑制作用调控。出乎意料的是，CAT8启动子内Mig1p结合基序的缺失并未增加CAT8的转录；此外，它导致CAT8启动子激活的丧失。使用启动子测试质粒的插入实验证实，这个20bp的调控元件也影响葡萄糖抑制和去抑制作用。这一发现表明该启动子区域具有上游激活功能，且不依赖于Mig1p，因为δmig1突变体仍能够使CAT8启动子去抑制。就葡萄糖调控的CAT8表达而言，其他假定的结合位点，如Hap2/3/4/5p位点和Abf1p共有位点均无功能。Cat8p与Gal4p DNA结合结构域的融合介导了转录激活。这种激活能力仍然受碳源调控，并依赖于Cat1p（Snf1p）蛋白激酶，这表明Cat8p需要翻译后修饰才能发挥其基因激活功能。事实上，十二烷基硫酸钠凝胶上的蛋白质印迹分析显示，葡萄糖培养细胞的粗提物中有一条带（Cat8pI），而去抑制细胞中鉴定出三条带（Cat8pI、-II和-III）。磷酸酶处理表明，去抑制特异性的Cat8pII和-III是由差异磷酸化产生的。只有磷酸化程度最高的修饰形式（Cat8pIII）依赖于Cat1p（Snf1p）激酶，这表明另一种蛋白激酶负责形成Cat8pII修饰。Cat8pIII的出现与糖异生酶（磷酸烯醇式丙酮酸羧激酶和果糖-1,6-二磷酸酶）和糖异生PCK1 mRNA的去抑制密切相关。此外，葡萄糖触发了Cat8pIII的去磷酸化，但这并不依赖于先前描述的参与蔗糖酶抑制的Glc7p（Cid1p）磷酸酶。这些结果证实了我们目前的模型，即糖异生基因的葡萄糖去抑制需要Cat8p磷酸化，并且还表明还涉及一个未知的转录激活因子。
