Analysis of phytochrome-dependent signal transduction in Aspergillus nidulans

真菌构巢曲霉和链格孢利用光敏色素来感知和响应红光。在光照15分钟后导致超过1000个基因差异表达的信号转导级联涉及HOG途径。在上一个资助期，转录变化被解开，光敏色素在远红光和蓝光传感中的新功能被发现。以. Alternata解决了关于线性四吡咯形成的长期存在的问题，并表征了两种血红素加氧酶。以.关于血红素开环的问题仍然没有解决。有趣的是，A. alternata HO与线粒体结合，在线粒体中它们直接与光敏色素脱辅基蛋白相互作用，直到其装载有发色团。现在，我们建议研究的C-末端的HoxA在线粒体膜协会的作用，并试图确定电子供体的HO。铁氧还蛋白和P450细胞色素是很好的候选者。在初步实验中，我们发现HoxB的C末端似乎也有一个功能，即阻止蛋白质进入细胞核。有效导入了截断版本。我们有证据表明，核功能与抗应激有关。血红素加氧酶HoxB在细胞核中的这种明显的新功能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短蛋白过表达后的转录组学反应来研究。最后，对A. alternata中的酶为鉴定A. nidulans。由于没有具有相似序列的蛋白质，我们预期一种非常独特的蛋白质。在上一个资助期，我们还建立了在大肠杆菌中生产大量光敏色素光敏结构域或全长蛋白的方法。杆菌这些蛋白质将用于光谱学和晶体学实验，与P. Hildebrandt和P.谢勒（柏林）合作。此外，光敏色素中特定氨基酸残基的作用将被测试其对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.