Function of a p24 Heterodimer in Morphogenesis and Protein Transport in Penicillium oxalicum

Abstract

The lignocellulose degradation capacity of filamentous fungi has been widely studied because of their cellulase hypersecretion. The p24 proteins in eukaryotes serve important functions in this secretory pathway. However, little is known about the functions of the p24 proteins in filamentous fungi. In this study, four p24 proteins were identified in Penicillium oxalicum. Six p24 double-deletion strains were constructed, and further studies were carried out with the ΔerpΔpδ strain. The experimental results suggested that Erp and Pδ form a p24 heterodimer in vivo. This p24 heterodimer participates in important morphogenetic events, including sporulation, hyphal growth, and lateral branching. The results suggested that the p24 heterodimer mediates protein transport, particularly that of cellobiohydrolase. Analysis of the intracellular proteome revealed that the ΔerpΔpδ double mutant is under secretion stress due to attempts to remove proteins that are jammed in the endomembrane system. These results suggest that the p24 heterodimer participates in morphogenesis and protein transport. Compared with P. oxalicum Δerp, a greater number of cellular physiological pathways were impaired in ΔerpΔpδ. This finding may provide new insights into the secretory pathways of filamentous fungi.

Figure 1. Erp and Pδ Homologs in P. oxalicum 114-2.

Panel a, phylogenetic tree of certain p24 proteins from eukaryotic species. Black rectangles represent PDE_09471, PDE_08336, PDE_00613 and PDE_03657. H, Homo sapiens; D, Drosophila melanogaster; S, Saccharomyces cerevisiae; X, Xenopus tropicalis; P, Pan troglodytes; A, Arabidopsis thaliana. Protein accession numbers are listed in Supplementary Information S1 online. Panel b, structural diagram of the p24 heterodimer, which is composed of Erp and Pδ. White represents Pδ, and black represents Erp. Each protein domain is marked with a bracket. The di-aromatic/large hydrophobic and di-basic motifs are highlighted with rectangles.

Figure 2. Visualisation of the Erp-Pδ Interaction Through the BiFC Assay.

Strains carrying a) eyfpN + eyfpC, b) eyfpN + pδ-eyfpC, c) erp-eyfpN + eyfpC, and d) erp-eyfpN + pδ-eyfpC. Scale bars: 10 μm.

Figure 3. Comparison of the Sporulation Efficiencies of the Parent and ΔerpΔpδ Strains.

*P < 0.05, **P < 0.01. Panel a, phenotypic analysis of the parent and ΔerpΔpδ strains on wheat bran plates. Panel b, the number of spores produced per square centimetre on wheat bran plates. Panel c, the number of spores produced per milligram of mycelial protein in submerged cultivation. Error bars indicate the standard deviation calculated from three biological experiments.

Figure 4. Comparison of Lateral Branching Development Patterns Between the Parent and ΔerpΔpδ Strains.

Panel a, microscopic observation of hyphae from the parent and ΔerpΔpδ strains. Scale bar: 10 μm. Panel b, statistical comparison of angle distribution between main hyphae and lateral branches in the parent and ΔerpΔpδ strains. White represents the parent strain, and black represents the ΔerpΔpδ strain. Error bars indicate the standard deviation calculated from three biological experiments.

Figure 5. Comparison of the Protein Secretion Capabilities of the Parent and ΔerpΔpδ Strains.

Panel a, comparison of extracellular protein concentrations and enzymatic activities between the parent and ΔerpΔpδ strains. Closed square, parent strain; open square, ΔerpΔpδ strain. Panel b, comparison of intracellular protein concentrations and specific enzymatic activities between the parent and ΔerpΔpδ strains. Closed square, parent strain; open square, ΔerpΔpδ strain. Error bars indicate the standard deviation calculated from three biological experiments.

Figure 6. Subcellular Distribution of Significantly Changed Proteins in Response to erp and pδ Deletions in P. oxalicum.