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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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Distinct subcellular localization patterns among melanin enzymes when constitutively expressed alone. (A) Colony images of the overexpression strains Ptef1-Alb1-GFP, Ptef1-Ayg1-GFP, Ptef1-Arp1-GFP, Ptef1-Arp2-GFP, Ptef1-Abr1-GFP, and Ptef1-Abr2-GFP. All strains produced DHN melanin during conidiation, as evidenced by the bluish grey conidial color that is similar to that of the wild-type colony. (B) Fluorescence images of vegetative hyphae of all the melanin enzyme overexpression strains. (C) Fluorescence images of conidia of all the melanin enzyme overexpression strains. (D) Relative transcript levels of the six melanin genes alb1, Ayg1, arp1, arp2, abr1, and abr2 in each of the melanin enzyme overexpression strains compared to that in WT during vegetative hyphal growth. Error bars show standard deviations. (E) Localization of the PKS enzyme Alb1-GFP and the endosomal marker Rab5-mCherry in vegetative hyphae (left), and a fluorescence intensity plot along a cellular axis indicated with a white line on the merged image (right). (F) Localization of the laccase Abr1-GFP and the endosomal marker Rab7-mCherry in vegetative hyphae (left), and a fluorescence intensity plot along a cellular axis indicated with a white line (right). The y axis used in panels E and F indicates the relative florescence units (RFUs).
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fig2: Distinct subcellular localization patterns among melanin enzymes when constitutively expressed alone. (A) Colony images of the overexpression strains Ptef1-Alb1-GFP, Ptef1-Ayg1-GFP, Ptef1-Arp1-GFP, Ptef1-Arp2-GFP, Ptef1-Abr1-GFP, and Ptef1-Abr2-GFP. All strains produced DHN melanin during conidiation, as evidenced by the bluish grey conidial color that is similar to that of the wild-type colony. (B) Fluorescence images of vegetative hyphae of all the melanin enzyme overexpression strains. (C) Fluorescence images of conidia of all the melanin enzyme overexpression strains. (D) Relative transcript levels of the six melanin genes alb1, Ayg1, arp1, arp2, abr1, and abr2 in each of the melanin enzyme overexpression strains compared to that in WT during vegetative hyphal growth. Error bars show standard deviations. (E) Localization of the PKS enzyme Alb1-GFP and the endosomal marker Rab5-mCherry in vegetative hyphae (left), and a fluorescence intensity plot along a cellular axis indicated with a white line on the merged image (right). (F) Localization of the laccase Abr1-GFP and the endosomal marker Rab7-mCherry in vegetative hyphae (left), and a fluorescence intensity plot along a cellular axis indicated with a white line (right). The y axis used in panels E and F indicates the relative florescence units (RFUs).

Mentions: As melanin enzymes are associated with protein complexes and early enzymes interact with late enzymes, it is tempting to speculate that the atypical secretory proteins (early enzymes) may piggyback onto the canonical secretory proteins (late enzymes) for their recruitment to the secretory pathway. If this piggyback hypothesis were valid, one would predict that each early melanin enzyme would become cytoplasmic when expressed alone. Because all melanin enzymes are coordinately upregulated during conidiation, it is challenging to examine the expression of any single melanin enzyme alone without the influence of other melanin enzymes. Hence, we took advantage of the fact that none of these enzymes were produced during vegetative hyphal growth (Fig. 1B), and we expressed each of the six GFP-tagged enzymes using the constitutively active tef1promoter. All six overexpression strains produced grayish blue melanin during conidiation, like the wild type (Fig. 2A), suggesting that overexpression of individual melanin enzymes did not impair melanization during conidiation. We noticed that overexpression of the PKS enzyme Alb1 led to the secretion of orange molecules into the medium during vegetative hyphal growth (Fig. 2A; see also Fig. S1 in the supplemental material). This was likely due to the ability of this polyketide synthase to supply precursors for multiple polyketides, as shown in Aspergillus niger (21). The constitutive expression of any one melanin gene did not show any significant impact on the transcript levels of other melanin genes during vegetative hyphal growth (Fig. 2D). During conidiation, all melanin genes were induced in these overexpression strains, as expected. Thus, these overexpression strains allowed us to examine the subcellular localization of individual melanin enzymes during vegetative hyphal growth without the interference of other melanin enzymes.


Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation
Distinct subcellular localization patterns among melanin enzymes when constitutively expressed alone. (A) Colony images of the overexpression strains Ptef1-Alb1-GFP, Ptef1-Ayg1-GFP, Ptef1-Arp1-GFP, Ptef1-Arp2-GFP, Ptef1-Abr1-GFP, and Ptef1-Abr2-GFP. All strains produced DHN melanin during conidiation, as evidenced by the bluish grey conidial color that is similar to that of the wild-type colony. (B) Fluorescence images of vegetative hyphae of all the melanin enzyme overexpression strains. (C) Fluorescence images of conidia of all the melanin enzyme overexpression strains. (D) Relative transcript levels of the six melanin genes alb1, Ayg1, arp1, arp2, abr1, and abr2 in each of the melanin enzyme overexpression strains compared to that in WT during vegetative hyphal growth. Error bars show standard deviations. (E) Localization of the PKS enzyme Alb1-GFP and the endosomal marker Rab5-mCherry in vegetative hyphae (left), and a fluorescence intensity plot along a cellular axis indicated with a white line on the merged image (right). (F) Localization of the laccase Abr1-GFP and the endosomal marker Rab7-mCherry in vegetative hyphae (left), and a fluorescence intensity plot along a cellular axis indicated with a white line (right). The y axis used in panels E and F indicates the relative florescence units (RFUs).
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fig2: Distinct subcellular localization patterns among melanin enzymes when constitutively expressed alone. (A) Colony images of the overexpression strains Ptef1-Alb1-GFP, Ptef1-Ayg1-GFP, Ptef1-Arp1-GFP, Ptef1-Arp2-GFP, Ptef1-Abr1-GFP, and Ptef1-Abr2-GFP. All strains produced DHN melanin during conidiation, as evidenced by the bluish grey conidial color that is similar to that of the wild-type colony. (B) Fluorescence images of vegetative hyphae of all the melanin enzyme overexpression strains. (C) Fluorescence images of conidia of all the melanin enzyme overexpression strains. (D) Relative transcript levels of the six melanin genes alb1, Ayg1, arp1, arp2, abr1, and abr2 in each of the melanin enzyme overexpression strains compared to that in WT during vegetative hyphal growth. Error bars show standard deviations. (E) Localization of the PKS enzyme Alb1-GFP and the endosomal marker Rab5-mCherry in vegetative hyphae (left), and a fluorescence intensity plot along a cellular axis indicated with a white line on the merged image (right). (F) Localization of the laccase Abr1-GFP and the endosomal marker Rab7-mCherry in vegetative hyphae (left), and a fluorescence intensity plot along a cellular axis indicated with a white line (right). The y axis used in panels E and F indicates the relative florescence units (RFUs).
Mentions: As melanin enzymes are associated with protein complexes and early enzymes interact with late enzymes, it is tempting to speculate that the atypical secretory proteins (early enzymes) may piggyback onto the canonical secretory proteins (late enzymes) for their recruitment to the secretory pathway. If this piggyback hypothesis were valid, one would predict that each early melanin enzyme would become cytoplasmic when expressed alone. Because all melanin enzymes are coordinately upregulated during conidiation, it is challenging to examine the expression of any single melanin enzyme alone without the influence of other melanin enzymes. Hence, we took advantage of the fact that none of these enzymes were produced during vegetative hyphal growth (Fig. 1B), and we expressed each of the six GFP-tagged enzymes using the constitutively active tef1promoter. All six overexpression strains produced grayish blue melanin during conidiation, like the wild type (Fig. 2A), suggesting that overexpression of individual melanin enzymes did not impair melanization during conidiation. We noticed that overexpression of the PKS enzyme Alb1 led to the secretion of orange molecules into the medium during vegetative hyphal growth (Fig. 2A; see also Fig. S1 in the supplemental material). This was likely due to the ability of this polyketide synthase to supply precursors for multiple polyketides, as shown in Aspergillus niger (21). The constitutive expression of any one melanin gene did not show any significant impact on the transcript levels of other melanin genes during vegetative hyphal growth (Fig. 2D). During conidiation, all melanin genes were induced in these overexpression strains, as expected. Thus, these overexpression strains allowed us to examine the subcellular localization of individual melanin enzymes during vegetative hyphal growth without the interference of other melanin enzymes.

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