Salmon Jemma, Ramos Jason, Callis Judy
Section of Molecular and Cellular Biology, University of California-Davis, 1 Shields Avenue, Davis, CA 95616, USA.
Plant J. 2008 Apr;54(1):118-28. doi: 10.1111/j.1365-313X.2007.03396.x. Epub 2007 Dec 15.
Auxin-mediated gene expression is largely controlled through a family of DNA-binding proteins known as auxin response factors (ARF). Previous studies on the role of proteolytic regulation in auxin signaling have focused on degradation of their interacting partner, the Aux/IAA proteins. Aux/IAA family members with domain II sequences are rapidly degraded, show auxin-enhanced degradation rates, and interact with the related F-box proteins TIR1 and AFB1-3, which indicates that they are ubiquitylated by a CUL1-dependent E3 ligase. To date, limited data have been generated regarding degradation of ARFs. Here, we focus on the degradation rate of one ARF family member, Arabidopsis thaliana ARF1, and find that the half-lives of N-terminally HA-tagged ARF1 and C-terminally luciferase-tagged ARF1 are both approximately 3-4 h. This half-life appears to be conferred by a component of the middle region (MR), and degradation of the luciferase fusion with the MR is more rapid when the fusion includes an additional nuclear localization signal. ARF1 degradation is proteasome-dependent and rates are not altered in a CUL1 mutant background, suggesting that this ARF is targeted for proteasomal degradation via an alternative set of machinery to that used for Aux/IAA degradation. Consistent with this, exogenous indole acetic acid does not affect the degradation of ARF1. Given increasing evidence that the relative ratio of Aux/IAAs to ARFs rather than the absolute quantity within the cell appears to be the mode through which auxin signaling is modulated, this half-life is likely to be biologically relevant.
生长素介导的基因表达在很大程度上是通过一类被称为生长素响应因子（ARF）的DNA结合蛋白来控制的。先前关于蛋白水解调控在生长素信号传导中作用的研究主要集中在其相互作用伴侣Aux/IAA蛋白的降解上。具有结构域II序列的Aux/IAA家族成员会迅速降解，显示出生长素增强的降解速率，并与相关的F-box蛋白TIR1和AFB1 - 3相互作用，这表明它们被一种依赖CUL1的E3连接酶泛素化。迄今为止，关于ARF降解的数据有限。在这里，我们聚焦于一个ARF家族成员——拟南芥ARF1的降解速率，发现N端带有HA标签的ARF1和C端带有荧光素酶标签的ARF1的半衰期均约为3 - 4小时。这种半衰期似乎是由中间区域（MR）的一个成分赋予的，当荧光素酶与MR的融合体包含额外的核定位信号时，其降解速度更快。ARF1的降解是蛋白酶体依赖性的，并且在CUL1突变背景下其降解速率没有改变，这表明该ARF是通过与用于Aux/IAA降解的另一套机制被靶向蛋白酶体降解的。与此一致的是，外源吲哚乙酸不会影响ARF1的降解。鉴于越来越多的证据表明，Aux/IAA与ARF的相对比例而非细胞内的绝对数量似乎是生长素信号传导被调节的方式，这种半衰期可能具有生物学意义。
