Bird A J, Zhao H, Luo H, Jensen L T, Srinivasan C, Evans-Galea M, Winge D R, Eide D J
Department of Nutritional Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA.
EMBO J. 2000 Jul 17;19(14):3704-13. doi: 10.1093/emboj/19.14.3704.
The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA-binding activity is conferred by five C-terminal C(2)H(2) zinc fingers and each finger is required for high-affinity DNA binding. The zinc-responsive domain of Zap1 also maps to the C-terminal zinc fingers. Furthermore, mutations that disrupt some of these fingers cause constitutive activity of a bifunctional Gal4 DNA-binding domain-Zap1 fusion protein. These results demonstrate a novel function of Zap1 zinc fingers in zinc sensing as well as DNA binding.
酿酒酵母的Zap1转录激活因子控制锌稳态。Zap1在锌限制的细胞中诱导靶基因表达，并受到高锌的抑制。其中一个靶基因就是Zap1自身。在本报告中，我们研究了锌如何调节Zap1的功能。首先，我们表明Zap1的转录自调控是锌反应性的一个次要组成部分；Zap1活性的大多数调节发生在翻译后。其次，Zap1的核定位不会因锌而改变，这表明锌调节DNA结合和/或激活域功能。为了了解Zap1如何对锌作出反应，我们对该蛋白质进行了功能剖析。Zap1包含两个激活域。DNA结合活性由五个C末端C(2)H(2)锌指赋予，每个锌指都是高亲和力DNA结合所必需的。Zap1的锌反应域也定位于C末端锌指。此外，破坏其中一些锌指的突变会导致双功能Gal4 DNA结合域-Zap1融合蛋白的组成型活性。这些结果证明了Zap1锌指在锌感应以及DNA结合中的新功能。
