Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin

Abstract

Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes.

Figure 1. Mutations in the endosomal sorting nexin Mvp1 cause lack of melanin deposition in the cell wall

(A) Colony images of WT, mvp1^Tn, and the melanin gene mutants (alb1Δ, ayg1Δ, arp1Δ, arp2Δ, abr1Δ & abr2Δ ). (B) SEM images of mature conidiophores of WT and the mvp1^Tn mutant. (C) SEM images of WT and mvp1^Tn conidia. (D) TEM images of intact conidia of WT, alb1Δ, abr1Δ, and mvp1^Tn mutants. White arrows point to the electron-dense melanin layer. Black arrows point to the electron-transparent cell wall layer. (E) TEM images of melanin ghosts extracted from WT and abr1Δ conidia. (F) Localization of Mvp1 and the endosomal marker Rab5. (G) Localization of Mvp1 and the endosomal marker Rab7. See also Figure S1.

Figure 2. Distinct localization patterns for the early and late melanin enzymes

(A) The melanin gene cluster in A. fumigatus and the predicted localization of melanin enzymes in diverse fungal species. S: Secreted protein; C: Cytosolic protein; PM: Plasma membrane; Mit: Mitochondria; ^a: Ascomycetes; ^b: Basidiomycetes; *: melanin genes not arranged in a cluster; ?: the presence of an ortholog is uncertain. (B) The expression and localization of Abr2 during development. [a] vegetative hypha; [b] young stalk; [c–d] mature stalk; [e–f] young conidiophore; [g] conidia. (C) Localization of Alb1 in conidia. (D) Localization of Alb1-GFP and the endosomal marker Rab5-mCherry, and a fluorescence intensity plot along a cellular axis indicated with a white line. (E) Localization of Alb1-GFP and Mvp1-mCherry, and a fluorescence intensity plot showing co-localization. Localization(F) and fluorescence intensity (G) of the early melanin enzyme wA-GFP in WT and mvp1Δ in A. nidulans. Localization (H) and fluorescence intensity (I) of the late melanin enzyme yA-chRFP in WT and mvp1Δ. Scale bars in panels D–F: 2.5 μm. See also Figure S2

Figure 3. Co-localization of two 14-3-3 isoforms, Bmh1 and Bmh2, with melanin enzymes Alb1 and Arp2

(A) Images of Alb1-GFP with either Bmh1-mCherry (top) or Bmh2-mCherry (bottom) in conidia. (B) The fluorescence intensity profiles of Alb1-GFP and Bmh1-mCherry plotted against the conidial cellular axis in the direction shown in the images at right. (C) Images of Arp2-GFP with either Bmh1-mCherry (top) or Bhm2-mCherry (bottom) in conidia. (D) The fluorescence intensity profiles of Arp2-GFP and Bmh2-mCherry plotted against the conidial cellular axis in the direction shown in the images at right.

Figure 4. The late enzymes accumulate in the cell wall

(A) Plasmolysis reveals the cell wall localization of Abr1 and intracellular localization of Mvp1. (B) Mechanic severing reveals Abr1’s association with the cell wall in the damaged stalk cell. Intracellular Mvp1 is retained in the intact conidia but is lost in the severed stalk cell. (C) Cells expressing Abr1-GFP and Abr2-mCherry were treated for plasmolysis. The separation of cytoplasm from the cell wall (DIC) reveals the localization of both Abr2 and Abr1 in the cell wall. Scale bar: 5 μm Localization (D) and fluorescence intensity (E) of Abr2-GFP in WT and abr1Δ (p <0.001). Scale bars: 5 μm. See also Figure S3