Red light-induced formation of ubiquitin-phytochrome conjugates: Identification of possible intermediates of phytochrome degradation.
Red light-induced formation of ubiquitin-phytochrome conjugates: Identification of possible intermediates of phytochrome degradation.
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DOI：
10.1073/pnas.84.2.359
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发表时间：
1987
影响因子：
11.1
通讯作者：
J. Shanklin;M. Jabben;R. Vierstra
中科院分区：
文献类型：
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作者：
J. Shanklin;M. Jabben;R. Vierstra
光敏色素是高等植物中控制红光介导的形态建成的光感受器。它以两种光互变形式存在，一种是红光吸收形式Pr，另一种是远红光吸收形式Pfr。由于Pr到Pfr的光转换通过一个简短的光脉冲降低了该色蛋白的体内半衰期约100倍，该系统提供了一种独特的方式来调节特定蛋白质的周转率，从而研究负责选择性蛋白质降解的机制。在黄化燕麦[Avena sativa（L.）幼苗中，光敏色素降解为Pfr遵循零级动力学，如光谱和通过ELISA测量的，在27 ℃下在约130分钟内损失50%的Pfr。抗燕麦光敏色素免疫球蛋白的破坏过程的免疫印迹分析揭示，降解涉及124 kDa光敏色素单体的损失，并且表观分子量低于124 kDa的蛋白水解中间体在体内不会积累到可检测的水平（<总光敏色素的0.015%）。后一种观察结果表明，蛋白质的蛋白水解分解非常迅速。然而，一系列的多肽具有较高的表观分子量和抗光敏色素免疫球蛋白（主要是129和134 kDa）的识别后，出现光转化为PFR。这些多肽代表不超过百分之几的总免疫可检测光敏色素池，并具有5 kDa的表观分子量的增量差异。它们在Pfr形成后5分钟内出现，在90和180分钟之间达到最高水平，此后下降。这些多肽和表观分子量高达160 kDa的其他多肽也可以用针对燕麦或人泛素的免疫球蛋白检测到，表明它们是泛素-光敏色素缀合物。由于泛素缀合参与细胞内蛋白质周转，并且由于Pfr-泛素缀合物的形成和降解与Pfr的周转一致，因此这些数据表明Pfr形式的光敏色素通过泛素依赖性蛋白水解途径降解。
Phytochrome is the photoreceptor that controls red light-mediated morphogenesis in higher plants. It exists in two photointerconvertible forms, a red light-absorbing form, Pr, and a far-red light-absorbing form, Pfr. Because photoconversion of Pr to Pfr by a brief light pulse decreases the in vivo half-life of this chromoprotein by a factor of approximately 100, this system offers a unique way to modulate the turnover rate of a specific protein and hence study the mechanisms responsible for selective protein degradation. In etiolated oat [Avena sativa (L.)] seedlings, degradation of phytochrome as Pfr follows zero-order kinetics as measured both spectrally and by ELISA, with 50% of Pfr lost in approximately 130 min at 27 degrees C. Immunoblot analysis of the destruction process with anti-oat phytochrome immunoglobulins reveals that degradation involves the loss of the 124-kDa phytochrome monomer and that proteolytic intermediates of apparent molecular mass lower than 124 kDa do not accumulate to detectable levels in vivo (<0.015% of total phytochrome). The latter observation suggests that proteolytic breakdown of the protein is extremely rapid. However, a series of polypeptides with higher apparent molecular mass and recognized by anti-phytochrome immunoglobulins (principally 129 and 134 kDa) appears after photoconversion to Pfr. These polypeptides represent no more than a few percent of the total immunologically detectable phytochrome pool and have incremental differences in apparent molecular mass of 5 kDa. They appear within 5 min after Pfr formation, reach maximal levels between 90 and 180 min, and decline thereafter. These polypeptides and others of apparent molecular mass up to 160 kDa are also detectable with immunoglobulins directed against either oat or human ubiquitin, indicating that they are ubiquitin-phytochrome conjugates. Since ubiquitin conjugation is involved in intracellular protein turnover and since formation and degradation of Pfr-ubiquitin conjugates coincide with the turnover of Pfr, these data suggest that the Pfr form of phytochrome is degraded via a ubiquitin-dependent proteolytic pathway.