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

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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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Detection of palmitoylated melanin enzymes in the overexpression strains. (A) A simplified flow chart for the experimental procedures to identify palmitoylated proteins. SH, thioester (free thiols); S-S-biotin, biotinylated disulfide. (B) Quantification of the ratios of recovered palmitoylated Alb1, Ayg1, Arp1, and Arp2 compared to the starting levels of the proteins was determined from total proteins extracted from vegetative hyphae of the Ptef1-alb1-GFP strain, the Ptef1-ayg1-GFP strain, the Ptef1-arp1-GFP strain, and the Ptef1-arp2-GFP strain. The Pmvp1-mvp1-GFP strain was used as a control. (B) GFP-tagged melanin enzymes extracted from vegetative hyphae of strains used in the Western blot analysis whose results are shown in panel C were detected with anti-GFP antibody in the total protein fraction (starting total protein prior to the in vitro processing), the +HA group (the palmitoylated protein pool), and the −HA group (negative control for palmitoylation protein pool). Mvp1-GFP was not detected in the palmitoylated protein group. (D) GFP-tagged melanin enzymes extracted from conidia of strains used in the Western blot analysis whose results are shown in panel E were detected with anti-GFP antibody in the total protein fraction (starting total protein prior to in vitro processing), the +HA group (the palmitoylated protein pool), and the −HA group (negative control for palmitoylation protein pool). Mvp1-GFP was again not detected in the palmitoylated protein group.
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fig5: Detection of palmitoylated melanin enzymes in the overexpression strains. (A) A simplified flow chart for the experimental procedures to identify palmitoylated proteins. SH, thioester (free thiols); S-S-biotin, biotinylated disulfide. (B) Quantification of the ratios of recovered palmitoylated Alb1, Ayg1, Arp1, and Arp2 compared to the starting levels of the proteins was determined from total proteins extracted from vegetative hyphae of the Ptef1-alb1-GFP strain, the Ptef1-ayg1-GFP strain, the Ptef1-arp1-GFP strain, and the Ptef1-arp2-GFP strain. The Pmvp1-mvp1-GFP strain was used as a control. (B) GFP-tagged melanin enzymes extracted from vegetative hyphae of strains used in the Western blot analysis whose results are shown in panel C were detected with anti-GFP antibody in the total protein fraction (starting total protein prior to the in vitro processing), the +HA group (the palmitoylated protein pool), and the −HA group (negative control for palmitoylation protein pool). Mvp1-GFP was not detected in the palmitoylated protein group. (D) GFP-tagged melanin enzymes extracted from conidia of strains used in the Western blot analysis whose results are shown in panel E were detected with anti-GFP antibody in the total protein fraction (starting total protein prior to in vitro processing), the +HA group (the palmitoylated protein pool), and the −HA group (negative control for palmitoylation protein pool). Mvp1-GFP was again not detected in the palmitoylated protein group.

Mentions: As in silico prediction could be unreliable, we decided to identify palmitoylated conidial proteins in the wild-type strain by using chemical reporters that mimic the natural lipids to label those proteins. Because these chemical reporters also contain biotin as a bio-orthogonal chemical handle that reacts with streptavidin (27), the originally palmitoylated proteins can then be purified through streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig. 5A). By this approach, we identified 234 palmitoylated proteins present in conidia, with 99 palmitoylated proteins detected at levels of 6 or more peptides per protein (see Table S1 in the supplemental material). Among these 99 palmitoylated proteins are several G-protein subunits and Rho GTPases that are known to be palmitoylated in other organisms (28, 29), RasA that is an experimentally verified palmitoylated protein in A. fumigatus and Cryptococcus neoformans (30, 31), and chaperon proteins that are enriched in previous palmitoylation proteomics studies (32–34) (see Table S1).


Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation
Detection of palmitoylated melanin enzymes in the overexpression strains. (A) A simplified flow chart for the experimental procedures to identify palmitoylated proteins. SH, thioester (free thiols); S-S-biotin, biotinylated disulfide. (B) Quantification of the ratios of recovered palmitoylated Alb1, Ayg1, Arp1, and Arp2 compared to the starting levels of the proteins was determined from total proteins extracted from vegetative hyphae of the Ptef1-alb1-GFP strain, the Ptef1-ayg1-GFP strain, the Ptef1-arp1-GFP strain, and the Ptef1-arp2-GFP strain. The Pmvp1-mvp1-GFP strain was used as a control. (B) GFP-tagged melanin enzymes extracted from vegetative hyphae of strains used in the Western blot analysis whose results are shown in panel C were detected with anti-GFP antibody in the total protein fraction (starting total protein prior to the in vitro processing), the +HA group (the palmitoylated protein pool), and the −HA group (negative control for palmitoylation protein pool). Mvp1-GFP was not detected in the palmitoylated protein group. (D) GFP-tagged melanin enzymes extracted from conidia of strains used in the Western blot analysis whose results are shown in panel E were detected with anti-GFP antibody in the total protein fraction (starting total protein prior to in vitro processing), the +HA group (the palmitoylated protein pool), and the −HA group (negative control for palmitoylation protein pool). Mvp1-GFP was again not detected in the palmitoylated protein group.
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fig5: Detection of palmitoylated melanin enzymes in the overexpression strains. (A) A simplified flow chart for the experimental procedures to identify palmitoylated proteins. SH, thioester (free thiols); S-S-biotin, biotinylated disulfide. (B) Quantification of the ratios of recovered palmitoylated Alb1, Ayg1, Arp1, and Arp2 compared to the starting levels of the proteins was determined from total proteins extracted from vegetative hyphae of the Ptef1-alb1-GFP strain, the Ptef1-ayg1-GFP strain, the Ptef1-arp1-GFP strain, and the Ptef1-arp2-GFP strain. The Pmvp1-mvp1-GFP strain was used as a control. (B) GFP-tagged melanin enzymes extracted from vegetative hyphae of strains used in the Western blot analysis whose results are shown in panel C were detected with anti-GFP antibody in the total protein fraction (starting total protein prior to the in vitro processing), the +HA group (the palmitoylated protein pool), and the −HA group (negative control for palmitoylation protein pool). Mvp1-GFP was not detected in the palmitoylated protein group. (D) GFP-tagged melanin enzymes extracted from conidia of strains used in the Western blot analysis whose results are shown in panel E were detected with anti-GFP antibody in the total protein fraction (starting total protein prior to in vitro processing), the +HA group (the palmitoylated protein pool), and the −HA group (negative control for palmitoylation protein pool). Mvp1-GFP was again not detected in the palmitoylated protein group.
Mentions: As in silico prediction could be unreliable, we decided to identify palmitoylated conidial proteins in the wild-type strain by using chemical reporters that mimic the natural lipids to label those proteins. Because these chemical reporters also contain biotin as a bio-orthogonal chemical handle that reacts with streptavidin (27), the originally palmitoylated proteins can then be purified through streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig. 5A). By this approach, we identified 234 palmitoylated proteins present in conidia, with 99 palmitoylated proteins detected at levels of 6 or more peptides per protein (see Table S1 in the supplemental material). Among these 99 palmitoylated proteins are several G-protein subunits and Rho GTPases that are known to be palmitoylated in other organisms (28, 29), RasA that is an experimentally verified palmitoylated protein in A. fumigatus and Cryptococcus neoformans (30, 31), and chaperon proteins that are enriched in previous palmitoylation proteomics studies (32–34) (see Table S1).

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