Tissue-specific and subcellular localization of phototropin determined by immuno-blotting
Tissue-specific and subcellular localization of phototropin determined by immuno-blotting
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DOI：
10.1007/s00425-003-1164-7
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发表时间：
2004-03-01
期刊：
影响因子：
4.3
通讯作者：
Rüdiger, W
中科院分区：
文献类型：
--
作者：
Knieb, E;Salomon, M;Rüdiger, W
向光素（Phototropin，phot）是一种紫外光/蓝光受体，介导植物对紫外光的向光反应.使用针对拟南芥phot 1的N-末端部分的抗血清，我们在这里展示了与来自燕麦、芸芥、大豆、独行菜、番茄、豌豆、白芥和玉米的向光素的交叉反应。在所有研究的植物中，蓝光照射导致对应于2-3 kDa的大小的明显增加的向光素的凝胶迁移率的转变。这种增长是短暂的：在60-90分钟内，在黑暗中向光蛋白带的表观大小恢复到原始大小。体外磷酸化的能力增加到350%（A. sativa）和200%（L. sativum），而不增加向光蛋白的量。从单子叶植物的胚芽鞘尖端，含有最高浓度的phototropin，我们发现一个指数下降，在基向节的大小相等，而线性下降，确定双子叶植物的基向节从下面的下胚轴钩。我们证实了在黑暗中生长的幼苗的所有phototropin的膜协会，2分钟的蓝光脉冲后，然而，20%的phototropin被发现在胞质组分和膜组分中只有80%。两种组分均显示凝胶迁移率变化，表明光依赖性自磷酸化。用黄化的A. sativa幼苗用溴化钠和二磷酸钠的混合物溶解高达95%的光致肽，随后使用Cibachron Blue 3GA-琼脂糖作为二核苷酸类似物进行亲和纯化。溶解后，可溶性phototropin仍然显示蓝光依赖性自磷酸化，但在不到1小时内失去活性。
Phototropin (phot) is a UV/blue- light receptor mediating phototropic reactions of plants as a response to unilateral irradiation. Using an antiserum directed against the N-terminal part of Arabidopsis phot1, we show here cross-reaction with phototropin from Avena sativa, Eruca sativa, Glycine max, Lepidium sativum, Lycopersicon esculentum, Pisum sativum, Sinapis alba, and Zea mays. In all investigated plants, blue light irradiation led to a gel mobility shift of phototropin corresponding to an apparent increase in size of 2-3 kDa. This increase is transient: the apparent size of the phototropin band reverted back to the original size in the dark within 60-90 min. The capacity for in vitro phosphorylation increased to 350% (A. sativa) and 200% (L. sativum) at 90 min after a blue light pulse without an increase in the amount of phototropin protein. Starting from coleoptile tips of monocots that contained the highest concentration of phototropin, we found an exponential decrease in basipetal sections of equal size while a linear decrease was determined for dicots in basipetal sections starting from the section below the hypocotyl hook. We confirmed the membrane association of all phototropin in dark-grown seedlings; after a 2-min blue light pulse, however, 20% of phototropin was found in the cytosolic fraction and only 80% in the membrane fraction. Both fractions showed the gel mobility shift indicating light-dependent autophosphorylation. Detergent-free solubilization of phototropin with chaotropic reagents was investigated with etiolated A. sativa seedlings. Up to 95% of phototropin was solubilized with a mixture of sodium bromide and sodium diphosphate, and subsequently subjected to affinity purification using Cibachron Blue 3GA-agarose as a dinucleotide analogue. Immediately after solubilization, soluble phototropin still showed blue-light-dependent autophosphorylation but lost its activity within less than 1 h.