Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis

Abstract

c 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.

Mutational analysis of the Aspergillus ambient pH receptor PalH underscores its potential as a target for antifungal compounds

“…Figure shows that in sharp contrast with the wild‐type, a proportion of phosphorylated PalH is detectable in fimA Δ cells under acidic conditions, strongly indicating that fimA Δ stabilizes this form. As fimA Δ prevents the endocytic internalization of PalH‐GFP (Lucena‐Agell et al ., ), this build‐up of the phosphorylated form suggests that phosphorylation tags the protein for endocytic turnover, a possibility that is consistent with the increase in the steady state levels of PalH detected in fimAΔ (and in palH654 ) cells.…”

“…With the single exception described below, Western blot analyses were made using ‘total protein samples’ obtained by a direct alkaline solubilization of lyophilized mycelium (Hervás‐Aguilar and Peñalva, ), which minimizes protein degradation and dephosphorylation. The sources of antibodies and the dilutions used for detection of Myc‐PacC, PalF‐HA3 (or PalH‐HA3), hexokinase and actin have been detailed previously (Lucena‐Agell et al ., ). Diagnostic tests of pH regulation based on alkaline pH (pH 8.3), lithium (0.3 or 0.5 M), sodium molybdate (10, 20 or 30 mM, as required) or neomycin sulfate (1 mg/ml) sensitivity have all been described (Tilburn et al ., ; Peñas et al ., ; Lucena‐Agell et al ., ).…”

Clathrin localization and dynamics in Aspergillus nidulans

“…The sole formin in A. nidulans , SepA, can be seen at the endocytic collar (our unpublished observations), and is important for hyphal growth (Harris et al ., ; Sharpless and Harris, ). However, the clathrin‐independent pathway in yeast was not dependent upon fimbrin, whereas FimA appears required for nearly all endocytosis (including FM4‐64 internalization) in A. nidulans (Upadhyay and Shaw, ; Lucena‐Agell et al ., ; Schultzhaus and Shaw, ).…”

Refining the pH response in Aspergillus nidulans: a modulatory triad involving PacX, a novel zinc binuclear cluster protein

“…pH signalling occurs at the plasma membrane in A. nidulans (Galindo et al ., ; Lucena‐Agell et al ., ) and S. cerevisiae (Obara and Kihara, ) where it involves dedicated Pal (Arst et al ., ) (or Rim) pH signal transduction components and the participation of certain endosomal sorting complex required for transport (ESCRT)‐I, ‐II and ‐III components (Xu et al ., ; Calcagno‐Pizarelli et al ., ; reviewed by Peñalva et al ., ). The plasma membrane sensor is PalH [Rim21 and Dgf16 in S. cerevisiae and C. albicans (Barwell et al ., ; Rothfels et al ., )].…”