Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells.
Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells.
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DOI：
10.1046/j.1365-313x.1997.11050967.x
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发表时间：
1997-05
期刊：
影响因子：
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通讯作者：
W. Terzaghi;R. Bertekap;A. Cashmore
中科院分区：
文献类型：
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作者：
W. Terzaghi;R. Bertekap;A. Cashmore
G-box是在许多不同基因的启动子中发现的重要调控元件。拟南芥基因家族的四个成员编码结合G-box的基本亮氨酸拉链蛋白（GBFs），此前已被克隆。为了研究GBFs，制备了一种针对细菌中表达的GBF1的多克隆抗体。该抗体也能识别GBF2和GBF3。对拟南芥和大豆（SB-M）细胞培养的细胞核和细胞质部分进行免疫印迹分析表明，90%以上的抗gbf1蛋白检测到的是细胞质。电泳迁移率转移试验表明，超过90%的G-box结合活性是细胞质的。DNA亲和层析表明，每个检测到抗gbf1蛋白特异性结合G-box。为了研究单个GBFs，制备了GBF1、GBF2和GBF4与GUS融合的DNA构建体，并通过在SB-M原生质体中的瞬时表达进行了检测。在所有测试条件下，50-62%的GUS:GBF1蛋白定位在细胞质中，而97%的GUS:GBF4蛋白定位在细胞核中。相比之下，在黑暗生长的细胞中，大约50%的GUS:GBF2蛋白存在于细胞质中，而在蓝光下培养的细胞中，超过80%的这种蛋白存在于细胞核中。GBF1的缺失分析发现，在氨基酸112和164之间有一个区域显然是细胞质保留所必需的。这些结果表明了一种有趣的可能性，即限制核通道可能是对GBF活动的重要控制。特别地，GBF2显然是在对光的反应中被特别输入细胞核的。
The G-box is an important regulatory element found in the promoters of many different genes. Four members of an Arabidopsis gene family encoding basic leucine zipper proteins (GBFs) which bind the G-box have previously been cloned. To study GBFs, a polyclonal antibody was raised against GBF1 expressed in bacteria. This antibody also recognized GBF2 and GBF3. Immunoblot analysis of nuclear and cytoplasmic fractions from Arabidopsis and soybean (SB-M) cell cultures indicated that over 90% of proteins detected with anti-GBF1 were cytoplasmic. Electrophoretic mobility shift assays indicated that over 90% of G-box binding activity was cytoplasmic. DNA affinity chromatography demonstrated that each protein detected with anti-GBF1 specifically bound the G-box. To study individual GBFs, DNA constructs fusing GBF1, GBF2 and GBF4 to GUS were made and assayed by transient expression in SB-M protoplasts. Of GUS:GBF1 proteins, 50-62% were localized in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 was localized in the nucleus. By contrast, whereas about 50% of GUS:GBF2 was found in the cytoplasm of dark-grown cells, over 80% of this protein was found in the nucleus in cells cultured under blue light. Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention. These results suggest the intriguing possibility that limitation of nuclear access may be an important control on GBF activity. In particular, GBF2 is apparently specifically imported into the nucleus in response to light.