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. 2015 Jun;14(6):545-53.
doi: 10.1128/EC.00031-15. Epub 2015 Apr 3.

Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis

Daniel Lucena-Agell  1 , Antonio Galindo  2 , Herbert N Arst Jr  3 , Miguel A Peñalva  4
Affiliations

Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis

Daniel Lucena-Agell et al. Eukaryot Cell. 2015 Jun.

Abstract

Aspergillus nidulans (Pal) ambient pH signaling takes place in cortical structures containing components of the ESCRT pathway, which are hijacked by the alkaline pH-activated, ubiquitin-modified version of the arrestin-like protein PalF and taken to the plasma membrane. There, ESCRTs scaffold the assembly of dedicated Pal proteins acting downstream. The molecular details of this pathway, which results in the two-step proteolytic processing of the transcription factor PacC, have received considerable attention due to the key role that it plays in fungal pathogenicity. While current evidence strongly indicates that the pH signaling role of ESCRT complexes is limited to plasma membrane-associated structures where PacC proteolysis would take place, the localization of the PalB protease, which almost certainly catalyzes the first and only pH-regulated proteolytic step, had not been investigated. In view of ESCRT participation, this formally leaves open the possibility that PalB activation requires endocytic internalization. As endocytosis is essential for hyphal growth, nonlethal endocytic mutations are predicted to cause an incomplete block. We used a SynA internalization assay to measure the extent to which any given mutation prevents endocytosis. We show that none of the tested mutations impairing endocytosis to different degrees, including slaB1, conditionally causing a complete block, have any effect on the activation of the pathway. We further show that PalB, like PalA and PalC, localizes to cortical structures in an alkaline pH-dependent manner. Therefore, signaling through the Pal pathway does not involve endocytosis.

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Figures

FIG 1
FIG 1
SynA as a reporter of endocytosis. (A) SynA is a single-pass v-SNARE delivered to the apex by exocytic carriers. Upon reaching the plasma membrane, it undergoes basipetal diffusion until captured by the subapical ring of endocytic patches (red circles), which acts as a diffusion barrier. (B) SynAen− is not taken up by endocytosis; therefore, it labels the plasma membrane uniformly (25). (C) In myoAS371E, endocytosis is accelerated (34).
FIG 2
FIG 2
Growth and SynA localization phenotypes of endocytosis mutants. (A) Growth tests (at 37°C) and GFP-SynA localization in germlings and hyphae in the sagAΔ mutant and in the wild type. (B) Growth phenotypes and SynA localization in fimAΔ and arfBΔ strains. (C) Growth and SynA localization phenotypes of slaB1 cells (22) cultured on ammonium (20 mM ammonium sulfate) or nitrate (10 mM sodium nitrate), which are strongly repressing and inducing conditions, respectively, for the niiAp-driven expression of SlaB. All microcopy images are displayed at the same magnification to facilitate interpanel comparisons.
FIG 3
FIG 3
Effect of endocytosis-impairing mutations on the localization of PalH-GFP. (A) Wild-type strain carrying a single-copy integration of a transgene driving the expression of PalH-GFP under the control of the moderately strong gpdAmini promoter (30). The boxed inset is shown at double magnification to better illustrate the faint GFP staining of the plasma membrane. Internal structures are endosomes and vacuoles. (B) sagAΔ strain expressing PalH-GFP; note the faint yet consistently greater staining of the plasma membrane compared to that of the wild-type control. (C) arfBΔ strain expressing PalH-GFP. Note the heterogeneous population consisting of cells with strong and moderate labeling of the plasma membrane, in both cases uniformly. The boxed hypha shows a cell of the moderate labeling and “more hyphal” class. (D) fimAΔ strain expressing PalH-GFP. Note the heterogeneous population, as in the case of the arfBΔ mutant. Fields in panels C and D are shown at half the magnification of those in panels A and B to illustrate population heterogeneity. As the gpdAmini::palH-gfp transgene was, in all cases, a single-copy integration of the construct targeted to the pyroA locus, levels of PalH-GFP expression are equivalent in all cases.
FIG 4
FIG 4
Proteolytic processing activation of PacC in null endocytic mutants. Wild-type and mutant cells expressing wild-type Myc-PacC72 from the gene replacement allele pacC900 were precultured under acidic conditions and shifted to alkaline conditions for the indicated times. Cells were collected and analyzed by Western blotting with anti-Myc antibody. Anti-hexokinase (hxk) was used as a loading control. (A) Wild-type versus myoAS371A cells. (B) Wild-type versus sagAΔ cells. (C) Wild-type versus fimAΔ cells. (D) Wild-type versus arfBΔ cells.
FIG 5
FIG 5
On ammonium, slaB1 completely prevents PalH endocytosis but does not alter the proteolytic processing activation of PacC. (A) slaB1 hyphae cultured on nitrate, expressing PalH-GFP as described in the legend to Fig. 3. (Left) Hyphae are localized to the plasma membrane at the nascent branch and predominate in the vacuoles (indicated by the letter “v”). (Right) Hyphal tip cells showing weak plasma membrane staining (arrows) and basal conidiospores showing large and conspicuously fluorescent vacuoles. (B) A large field at the same magnification as those used for panel A showing the homogenous population of yeast-like cells resulting from cultivating the slaB1 mutant on ammonium. The PalH-GFP puncta noticeable at the plasma membrane are large pits (not shown). (C) Western blot analysis of SlaB in slaB1 cells cultured on ammonium and shifted from acidic to alkaline pH. The anti-SlaB Western blots in the top and middle panels represent two different exposures (exp) for the same experiment. The lower panel is an anti-hexokinase (hxk) loading control. The middle panel was deliberately overexposed to reveal traces of SlaB. (D) Normal proteolytic processing activation of PacC in slaB1 cells, cultured on ammonium or nitrate, compared to the wild type. As for panel C, the anti-SlaB blot was deliberately overexposed to illustrate the extent of downregulation. Nitrate conditions result in marked overexpression of SlaB.
FIG 6
FIG 6
PalB is recruited to cortical puncta in an alkaline-dependent manner. (A) Diagnostic tests of pH regulation for palB-GFP. The null palB38 mutation prevents growth at pH 8.3 or on 0.3 M LiCl-containing media, and it leads to hypersensitivity to 10 mM sodium molybdate and to resistance to 1 mg/ml of neomycin (Neo). A palB-GFP gene replacement strain grows like the wild type at pH 8.3 and on LiCl and sodium molybdate plates, and it is as sensitive as the wild type to neomycin, indicating that tagging does not impair function. (B) Western blot analysis of cells expressing endogenously tagged PalA-GFP or PalB-GFP. Actin is a loading control. (C) Cells expressing PalB-GFP under the control of alcAp were cultured on ethanol medium at acidic pH and shifted to acidic (H+; pH 5.2) or alkaline (OH−; pH 8.2) pH for 30 min before being photographed. The diagram on the right is a quantitation of the number of cortical structures per micron counted in 32 hyphal tip cells cultured under acidic or alkaline conditions. Error bars are standard errors. The two sets of measurements are significantly different (P < 0.001) as determined with the Mann-Whitney U test.
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