Proper folding of the antifungal protein PAF is required for optimal activity

The Penicillium chrysogenum antifungal 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 PAR nor on the function of its prosequence in protein activity. Therefore. we to investigate the effects of secreted PAF as well as of intracellularly retained pro-PAF and mature PAF on the sensitive ascomycete Aspergillus nidulans, paf-coding sequences under the control and transformed this model organism by expression vectors containing 5'-sequentially truncated paf 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 RAF. 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 mitrochondrial 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 PAR The results give evidence that properly folded PAF is a prerequisite for its activity. (C) 2004 Elsevier SAS. All rights reserved.