Analysis of phytochrome-dependent signal transduction in Aspergillus nidulans

灰曲霉和互交霉利用光敏色素来感知和响应红光。光照15分钟后导致1000多个基因差异表达的信号转导级联涉及HOG途径。在之前的资助期内，揭示了转录变化，并发现了光敏色素在远红光和蓝光传感中的新功能。解决了长时间存在的关于线性四吡咯形成的问题，并对两种血红酶进行了表征。在毛竹中，血红素环打开的问题仍未得到解决。有趣的是，这两种草属植物的HOs都与线粒体相关，在线粒体中它们直接与光敏色素载脂蛋白相互作用，直到光敏色素载脂蛋白被装载到发色团中。现在我们打算研究HoxA的c端在线粒体膜结合中的作用，并试图确定HoxA的电子供体。铁氧化还原蛋白和P450细胞色素是很好的候选物质。在初步实验中，我们发现HoxB的c端似乎也有一个功能，即阻止蛋白质进入细胞核。有效地导入了删节版。我们有证据表明核功能与抗逆性有关。研究人员将通过分析蛋白质与“应激”转录因子的相互作用，以及研究截断蛋白过表达后的转录组反应，来研究HoxB在细胞核中的这种明显的新功能。最后，互花草的HO突变体提供了在空心草中鉴定相应酶的可能性。由于没有具有相似序列的蛋白质，我们预计是一种非常独特的蛋白质。在之前的资助期内，我们还建立了在大肠杆菌中生产大量光敏色素光感结构域或全长蛋白的方法。这些蛋白质将用于光谱和晶体学实验，与P. Hildebrandt和P. Scherer（柏林）合作。此外，将测试光敏色素中特定氨基酸残基对其功能和构象变化的贡献。

The fungi Aspergillus nidulans and Alternaria alternata use phytochrome to sense and respond to red light. The signal transduction cascade which leads to differential expression of more than 1000 genes after 15 min of illumination involves the HOG pathway. In the previous funding period, the transcriptional changes were unraveled, and novel functions of phytochrome in far-red and in blue-light sensing were discovered. In A. alternata a long-standing question about the formation of the linear tetrapyrrole was solved and two hemeoxyenases characterized. In A. nidulans the question about heme ring opening is still unsolved. Interestingly, both A. alternata HOs associated to mitochondria where they directly interact with the phytochrome apoprotein until this is loaded with the chromophore. Now we propose to study the role of the C-terminus of HoxA in mitochondrial membrane association and to try to identify the electron donor for the HOs. Ferredoxin and a P450 cytochrome are good candidates. In preliminary experiments, we found that the C-terminus of HoxB appears to also serve a function, namely, to prevent the protein from moving into nuclei. A truncated version was imported efficiently. We have evidence that the nuclear function is related to stress resistance. This apparently novel function of hemeoxygenase HoxB in nuclei will be studied through the analysis of protein interactions with “stress”-transcription factors as well as the study of a transcriptomic response after overexpression of the truncated protein. Lastly, the HO mutants in A. alternata offer the possibility to identify a corresponding enzyme in A. nidulans. Since there are no proteins with similar sequences, we anticipate a very distinct protein. In the previous funding period, we established also methods to produce large amounts of the phytochrome photosensory domain or the full-length protein in E. coli. These proteins will be used for spectroscopic and crystallographic experiments, in collaboration with P. Hildebrandt and P. Scherer (Berlin). In addition, the role of specific amino acid residues in phytochrome will be tested for their contribution to function and conformational changes.