Limits...
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.

No MeSH data available.


Related in: MedlinePlus

Constitutively expressed melanin enzymes are associated with protein complexes during conidiation but not during vegetative hyphal growth. FPLC profiles of the constitutively expressed melanin enzymes during vegetative hyphal growth (right) and during conidiation (left) are shown. The FPLC profiles are of Ptef1-alb1-GFP, Ptef1-ayg1-GFP, Ptef1-arp1-GFP, Ptef1-arp2-GFP, Ptef1-abr1-GFP, and Ptef1-abr2-GFP strains. The standard marker protein profiles (same as in Fig. 1D) are included here as a reference. The elution volumes used for graphing all the x axes are identical. The y axis indicates the relative florescence units (RFUs).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120144&req=5

fig3: Constitutively expressed melanin enzymes are associated with protein complexes during conidiation but not during vegetative hyphal growth. FPLC profiles of the constitutively expressed melanin enzymes during vegetative hyphal growth (right) and during conidiation (left) are shown. The FPLC profiles are of Ptef1-alb1-GFP, Ptef1-ayg1-GFP, Ptef1-arp1-GFP, Ptef1-arp2-GFP, Ptef1-abr1-GFP, and Ptef1-abr2-GFP strains. The standard marker protein profiles (same as in Fig. 1D) are included here as a reference. The elution volumes used for graphing all the x axes are identical. The y axis indicates the relative florescence units (RFUs).

Mentions: To examine whether the recruitment of these enzymes to the secretory pathway correlates with their association with protein complexes, we performed size exclusion chromatography for proteins extracted from vegetative hyphae and from young conidia of the six overexpression strains. None of the melanin enzymes formed detectable complexes when overexpressed alone in vegetative hyphae, based on the absence of GFP signals in fractions corresponding to sizes larger than those of each respective single protein alone (Fig. 3, right, peaks b1 to b6 represent the single proteins). In contrast, all six enzymes formed complexes in conidia, based on the presence of GFP signals in fractions corresponding to sizes much larger than those of each respective single protein alone (Fig. 3, left, peaks a1 to a6). The observation confirms our earlier results showing that the PKS enzyme Alb1 and the two conventional secretory proteins Abr1 and Abr2 could be sorted to the secretory pathway independently of other melanin enzymes.


Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation
Constitutively expressed melanin enzymes are associated with protein complexes during conidiation but not during vegetative hyphal growth. FPLC profiles of the constitutively expressed melanin enzymes during vegetative hyphal growth (right) and during conidiation (left) are shown. The FPLC profiles are of Ptef1-alb1-GFP, Ptef1-ayg1-GFP, Ptef1-arp1-GFP, Ptef1-arp2-GFP, Ptef1-abr1-GFP, and Ptef1-abr2-GFP strains. The standard marker protein profiles (same as in Fig. 1D) are included here as a reference. The elution volumes used for graphing all the x axes are identical. The y axis indicates the relative florescence units (RFUs).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Constitutively expressed melanin enzymes are associated with protein complexes during conidiation but not during vegetative hyphal growth. FPLC profiles of the constitutively expressed melanin enzymes during vegetative hyphal growth (right) and during conidiation (left) are shown. The FPLC profiles are of Ptef1-alb1-GFP, Ptef1-ayg1-GFP, Ptef1-arp1-GFP, Ptef1-arp2-GFP, Ptef1-abr1-GFP, and Ptef1-abr2-GFP strains. The standard marker protein profiles (same as in Fig. 1D) are included here as a reference. The elution volumes used for graphing all the x axes are identical. The y axis indicates the relative florescence units (RFUs).
Mentions: To examine whether the recruitment of these enzymes to the secretory pathway correlates with their association with protein complexes, we performed size exclusion chromatography for proteins extracted from vegetative hyphae and from young conidia of the six overexpression strains. None of the melanin enzymes formed detectable complexes when overexpressed alone in vegetative hyphae, based on the absence of GFP signals in fractions corresponding to sizes larger than those of each respective single protein alone (Fig. 3, right, peaks b1 to b6 represent the single proteins). In contrast, all six enzymes formed complexes in conidia, based on the presence of GFP signals in fractions corresponding to sizes much larger than those of each respective single protein alone (Fig. 3, left, peaks a1 to a6). The observation confirms our earlier results showing that the PKS enzyme Alb1 and the two conventional secretory proteins Abr1 and Abr2 could be sorted to the secretory pathway independently 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