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Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

View Article: PubMed Central - PubMed

ABSTRACT

Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.

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The subcellular localization of the other early melanin enzymes, Ayg1 and Arp1, is independent of that of the PKS enzyme Alb1. (A and B) Localization of constitutively expressed Ayg1-GFP and Arp1-GFP in vegetative hyphae (A) and conidia (B) in the alb1Δ mutant background. Scale bar, 5 µm. (C) The relative transcript levels of ayg1, arp1, and arp2 in the wild type and the alb1Δ mutant following induction during conidiation (24 h after transfer to solid medium). The transcript level of each gene was compared to that of the wild type during vegetative hyphal growth. Error bars show standard deviations.
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fig4: The subcellular localization of the other early melanin enzymes, Ayg1 and Arp1, is independent of that of the PKS enzyme Alb1. (A and B) Localization of constitutively expressed Ayg1-GFP and Arp1-GFP in vegetative hyphae (A) and conidia (B) in the alb1Δ mutant background. Scale bar, 5 µm. (C) The relative transcript levels of ayg1, arp1, and arp2 in the wild type and the alb1Δ mutant following induction during conidiation (24 h after transfer to solid medium). The transcript level of each gene was compared to that of the wild type during vegetative hyphal growth. Error bars show standard deviations.

Mentions: The polyketide synthase Alb1 colocalizes with the remaining early enzymes in secretory endosomes (19), and this PKS can be recruited to the secretory pathway independently of other melanin enzymes. To test whether Alb1 helps recruit the other atypical early melanin enzymes, we decided to examine the subcellular localization of Ayg1 in the absence of the alb1 gene. The deletion of the alb1 gene did not impair the induction of expression of the ayg1, arp1, and arp2 genes during conidiation (Fig. 4C). Overexpressed Ayg1 in the alb1Δ background, however, showed the same subcellular localization as Ayg1 in the wild-type background in both vegetative hyphae (Fig. 4A and 2B) and conidia (Fig. 4B and 2C). Similarly, the deletion of alb1 did not affect the subcellular localization of Arp1-GFP (Fig. 4B). Thus, the trafficking of other early melanin enzymes does not depend on Alb1.


Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation
The subcellular localization of the other early melanin enzymes, Ayg1 and Arp1, is independent of that of the PKS enzyme Alb1. (A and B) Localization of constitutively expressed Ayg1-GFP and Arp1-GFP in vegetative hyphae (A) and conidia (B) in the alb1Δ mutant background. Scale bar, 5 µm. (C) The relative transcript levels of ayg1, arp1, and arp2 in the wild type and the alb1Δ mutant following induction during conidiation (24 h after transfer to solid medium). The transcript level of each gene was compared to that of the wild type during vegetative hyphal growth. Error bars show standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120144&req=5

fig4: The subcellular localization of the other early melanin enzymes, Ayg1 and Arp1, is independent of that of the PKS enzyme Alb1. (A and B) Localization of constitutively expressed Ayg1-GFP and Arp1-GFP in vegetative hyphae (A) and conidia (B) in the alb1Δ mutant background. Scale bar, 5 µm. (C) The relative transcript levels of ayg1, arp1, and arp2 in the wild type and the alb1Δ mutant following induction during conidiation (24 h after transfer to solid medium). The transcript level of each gene was compared to that of the wild type during vegetative hyphal growth. Error bars show standard deviations.
Mentions: The polyketide synthase Alb1 colocalizes with the remaining early enzymes in secretory endosomes (19), and this PKS can be recruited to the secretory pathway independently of other melanin enzymes. To test whether Alb1 helps recruit the other atypical early melanin enzymes, we decided to examine the subcellular localization of Ayg1 in the absence of the alb1 gene. The deletion of the alb1 gene did not impair the induction of expression of the ayg1, arp1, and arp2 genes during conidiation (Fig. 4C). Overexpressed Ayg1 in the alb1Δ background, however, showed the same subcellular localization as Ayg1 in the wild-type background in both vegetative hyphae (Fig. 4A and 2B) and conidia (Fig. 4B and 2C). Similarly, the deletion of alb1 did not affect the subcellular localization of Arp1-GFP (Fig. 4B). Thus, the trafficking of other early melanin enzymes does not depend on Alb1.

View Article: PubMed Central - PubMed

ABSTRACT

Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.

No MeSH data available.


Related in: MedlinePlus