Figure 1.
Subcellular localization of Pex11 family members.
P. chrysogenum cells producing C-terminal mGFP fusions of Pex11 (A), Pex11C (B) or Pex11B (C) with DsRed.SKL as peroxisome marker were grown for 40 h in PPM and analyzed by CLSM. D. CLSM analysis of P. chrysogenum hyphae producing Pex11B.mGFP and Sec63.mCherry as marker of the ER, grown for 40 h in PPM. Scale bars represent 5 µm. Arrowheads (in C) indicate the sites of overlap between Pex11B.mGFP and Sec63.mCherry fluorescence.
Figure 2.
DsRed.SKL labeled peroxisomes co-localize with Pex11B.mGFP tagged ER at peroxisome inducing conditions in P. chrysogenum apical cells.
P. chrysogenum cells producing Pex11B.mGFP and DsRed.SKL were grown in PPM with addition of oleic acid (0.1%) and analyzed by CLSM. Co-localization of red fluorescent peroxisomes with Pex11B.mGFP labeled ER was observed in hyphal tips. The frequency of these events decreased towards the older subapical cells. The scale bar represents 5 µm.
Figure 3.
Impact of deletion of individual Pex11 family genes on peroxisome numbers.
P. chrysogenum hdfA GFP.SKL (A), Δpex11 GFP.SKL (B), Δpex11B GFP.SKL (C), Δpex11C GFP.SKL (D) cells were grown for 40 h on PPM and analyzed by CLSM. Scale bars represent 5 µm. E. Quantification of peroxisome numbers in P. chrysogenum cells depleted of individual pex11 family genes; n.s.- statistically not significant based on student t test. Error bars represent SEM.
Figure 4.
The effect of overproduction of Pex11 family members on peroxisome proliferation.
Representative CLSM images of P. chrysogenum GFP.SKL cells: WT (A); overproducing Pex11 (B), Pex11B (C) or Pex11C (D). Cells were grown for 40 h in PPM. Scale bars represent 5 µm. E. Western blot showing overproduction of Pex11 (top), Pex11B, (center) or Pex11C (bottom). Crude extracts of DS17690 and strains overproducing Pex11 proteins were used for SDS-PAGE and western blotting and probed with specific antisera. Equal amounts of protein were loaded per lane. F. Electron micrograph of P. chrysogenum cells overproducing Pex11B. P-peroxisome; M-mitochondrion. Scale bar represents 1 µm.
Figure 5.
P. chrysogenum cells devoid of all Pex11 family members still contain peroxisomes.
Double and triple deletion mutants of pex11 family genes were prepared and analyzed by CLSM after growth for 40 h in PPM: WT (A), Δpex11 (B), Δpex11 Δpex11B GFP.SKL (C), Δpex11 Δpex11C GFP.SKL (D), Δpex11B Δpex11C GFP.SKL (E), Δpex11 Δpex11B Δpex11C GFP.SKL (F). Scale bars represent 5 µm.
Figure 6.
P. chrysogenum Pex16 is a peroxisomal membrane protein.
P. chrysogenum cells producing Pex16.mGFP and either DsRed.SKL (A) or Sec63.mCherry (B) were grown for 40 h in PPM and analyzed by fluorescence microscopy. Scale bars represent 5 µm.
Figure 7.
Pex16 is not essential for peroxisome biogenesis.
Fluorescence microscopy analysis of P. chrysogenum Δpex16 GFP.SKL (A) and Δpex11 Δpex11B Δpex11C Δpex16 GFP.SKL (B) cells. Cells were grown for 40 h in PPM. For corresponding WT see Fig. 3A. Scale bars represent 5 µm. C. Cells devoid of pex16 are sporulation deficient. Colonies of WT and both Δpex16 GFP.SKL and Δpex11 Δpex11B Δpex11C Δpex16 GFP.SKL strains were grown for 7 days on sporulation inducing R-agar plates. D. Δpex16 cells are characterized by decreased levels of Pex11. Western blots of WT and Δpex16 GFP.SKL cell extracts were prepared and decorated with α-Pex11 antibodies; translation elongation factor 1- (eEF1A) was used as a loading control. E. P. chrysogenum cells lacking pex16 are able to grow on oleic acid although at decreased rates. WT, Δpex16 GFP.SKL and Δpex11 Δpex11B Δpex11C Δpex16 GFP.SKL strains were grown for 10 days on mineral medium containing 0.5% glucose or 0.1% oleic acid as a sole carbon source. Electron micrographs of Δpex16 GFP.SKL (F) and Δpex11 Δpex11B Δpex11C Δpex16 GFP.SKL (G) cells grown for 40 h in PPM; P – peroxisome; M – mitochondrion; V – vacuole; arrows indicate protein dense inclusions. Scale bars represent 1 µm. Electron micrographs representing α-IAT immunolabelling of sections of Δpex16 GFP.SKL (H) and Δpex11 Δpex11B Δpex11C Δpex16 GFP.SKL (I) cells. Scale bars represent 1 µm.
Figure 8.
Deletion of pex3 in P. chrysogenum results in cells completely devoid of peroxisomes.
P. chrysogenum Δpex3 cells, producing GFP.SKL were grown for 40 h in PPM medium and analyzed by fluorescence (A) and electron (B) microscopy. Scale bars represent 5 µm in A and 1 µm in B; M-mitochondrion, N – nucleus.
Figure 9.
Impact of deletion of pex11 family genes, pex16 or pex3 on penicillin production.
A. Analysis of the production of antibacterial compounds by selected strains using plate bioassays with M. luteus as an indicator strain. Clarified culture supernatants were diluted 3200 times before analysis. During each experiment a corresponding WT supernatant at the same dilution was tested on the same plate as the supernatants of the analyzed mutants. B and C. Western blot analysis of the levels of penicillin biosynthetic enzymes IPNS and IAT in strains with manipulated levels of Pex11 family proteins (B), Pex3 or Pex16 (C). eEF1A was used as a loading control.