Marx Florentine, Salvenmoser Willibald, Kaiserer Lydia, Graessle Stefan, Weiler-Görz Renate, Zadra Ivo, Oberparleiter Christoph
Department of Molecular Biology, Medical University of Innsbruck, Peter-Mayr Strasse 4B/III, 6020 Innsbruck, Austria.
Res Microbiol. 2005 Jan-Feb;156(1):35-46. doi: 10.1016/j.resmic.2004.07.007.
The Penicillium chrysogenumantifungal protein PAF is secreted into the supernatant after elimination of a preprosequence. PAF is actively internalized into the hyphae of sensitive molds and provokes growth retardation as well as changes in morphology. Thus far, no information is available on the exact mode of action of PAF, nor on the function of its prosequence in protein activity. Therefore, we sought to investigate the effects of secreted PAF as well as of intracellularly retained pro-PAF and mature PAF on the sensitive ascomycete Aspergillus nidulans, and transformed this model organism by expression vectors containing 5'-sequentially truncated paf-coding sequences under the control of the inducible P. chrysogenum-derived xylanase promoter. Indirect immunofluorescence staining revealed the localization of recombinant PAF predominantly in the hyphal tips of the transformant Xylpaf1 which expressed prepro-PAF, whereas the protein was found to be distributed intracellularly within all segments of hyphae of the transformants Xylpaf2 and Xylpaf3 which expressed pro-PAF and mature PAF, respectively. Growth retardation of Xylpaf1 and Xylpaf3 hyphae was detected by proliferation assays and by light microscopy analysis. Using transmission electron microscopy of ultrathin hyphal sections a marked alteration of the mitochondrial ultrastructure in Xylpaf1 was observed and an elevated amount of carbonylated proteins pointed to severe oxidative stress in this strain. The effects induced by secreted recombinant PAF resembled those evoked by native PAF. The results give evidence that properly folded PAF is a prerequisite for its activity.
产黄青霉抗真菌蛋白PAF在去除前导序列后分泌到上清液中。PAF可被主动内化到敏感霉菌的菌丝中，导致生长迟缓以及形态变化。到目前为止，关于PAF的确切作用方式及其前导序列在蛋白质活性中的功能尚无信息。因此，我们试图研究分泌型PAF以及细胞内保留的前体PAF和成熟PAF对敏感子囊菌构巢曲霉的影响，并通过含有在诱导型产黄青霉来源的木聚糖酶启动子控制下5'-顺序截短的paf编码序列的表达载体转化这种模式生物。间接免疫荧光染色显示重组PAF主要定位于表达前体PAF的转化体Xylpaf1的菌丝尖端，而在分别表达前体PAF和成熟PAF的转化体Xylpaf2和Xylpaf3的菌丝所有区段内发现该蛋白分布在细胞内。通过增殖试验和光学显微镜分析检测到Xylpaf1和Xylpaf3菌丝的生长迟缓。使用超薄菌丝切片的透射电子显微镜观察到Xylpaf1中线粒体超微结构有明显改变，并且羰基化蛋白量的增加表明该菌株存在严重的氧化应激。分泌的重组PAF诱导的效应类似于天然PAF引起的效应。结果表明正确折叠的PAF是其活性的先决条件。
