Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway
Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway
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DOI：
10.1074/jbc.m110.135236
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发表时间：
2010-07-09
影响因子：
4.8
通讯作者：
Dyer, John M.
中科院分区：
文献类型：
--
作者：
O'Quin, Jami B.;Bourassa, Linda;Dyer, John M.
环境温度的变化对变温生物体的膜构成了重大的生理挑战。在植物中，内质网（ER）定位的ω-3脂肪酸去饱和酶（FAD 3）在较冷的温度下增加多不饱和脂肪酸的产生，但FAD 3基因本身在这种适应性反应期间通常不会上调。在这里，我们在酵母细胞中表达了两个密切相关的植物FAD 3基因，发现它们的酶产生了显著不同数量的omega-3脂肪酸，这些差异与蛋白质周转率的差异相关。结构域交换和诱变实验表明，每种蛋白质在其N末端含有降解信号，并且该区域内的PEST样序列的电荷密度在很大程度上是蛋白质周转率差异的原因。每种Fad 3蛋白的半衰期在较冷的温度下增加，并且蛋白质降解需要ER相关降解途径的特定组分，包括Cdc 48衔接蛋白Doa 1、Shp 1和Ufd 2。Fad 3蛋白在与蛋白酶体抑制剂MG 132孵育的烟草细胞中的表达进一步证实了它们在植物中通过蛋白酶体途径降解。总的来说，这些研究结果表明，Fad 3蛋白丰度的顺式作用的降解信号和泛素-蛋白酶体途径的组合调节和Fad 3蛋白量的调制响应于温度可能代表一种机制的同源粘性适应植物。
Changes in ambient temperature represent a major physiological challenge to membranes of poikilothermic organisms. In plants, the endoplasmic reticulum (ER)-localized omega-3 fatty-acid desaturases (Fad3) increase the production of polyunsaturated fatty acids at cooler temperatures, but the FAD3 genes themselves are typically not up-regulated during this adaptive response. Here, we expressed two closely related plant FAD3 genes in yeast cells and found that their enzymes produced significantly different amounts of omega-3 fatty acids and that these differences correlated to differences in rates of protein turnover. Domain-swapping and mutagenesis experiments revealed that each protein contained a degradation signal in its N terminus and that the charge density of a PEST-like sequence within this region was largely responsible for the differences in rates of protein turnover. The half-life of each Fad3 protein was increased at cooler temperatures, and protein degradation required specific components of the ER-associated degradation pathway including the Cdc48 adaptor proteins Doa1, Shp1, and Ufd2. Expression of the Fad3 proteins in tobacco cells incubated with the proteasomal inhibitor MG132 further confirmed that they were degraded via the proteasomal pathway in plants. Collectively, these findings indicate that Fad3 protein abundance is regulated by a combination of cis-acting degradation signals and the ubiquitin-proteasome pathway and that modulation of Fad3 protein amounts in response to temperature may represent one mechanism of homeoviscous adaptation in plants.