title: An ordered pathway for the assembly of fungal ESCRT-containing ambient pH signalling complexes at the plasma membrane.
abstract: The fungal pal/RIM signalling pathway, which regulates gene expression in response to environmental pH involves, in addition to dedicated proteins, several components of ESCRT complexes, which suggested that pH signalling proteins assemble on endosomal platforms. In Aspergillus nidulans, dedicated Pal proteins include the plasma membrane receptor PalH and its coupled arrestin, PalF, which becomes ubiquitylated in alkaline pH conditions, and three potentially endosomal ESCRT-III associates, including Vps32 interactors PalA and PalC and Vps24 interactor calpain-like PalB. We studied the subcellular locations at which signalling takes place after activating the pathway by shifting ambient pH to alkalinity. Rather than localising to endosomes, Vps32 interactors PalA and PalC transiently colocalise at alkaline-pH-induced cortical structures in a PalH-, Vps23- and Vps32-dependent but Vps27-independent manner. These cortical structures are much more stable when Vps4 is deficient, indicating that their half-life depends on ESCRT-III disassembly. Pull-down studies revealed that Vps23 interacts strongly with PalF, but co-immunoprecipitates exclusively with ubiquitylated PalF forms from extracts. We demonstrate that Vps23-GFP, expressed at physiological levels, is also recruited to cortical structures, very conspicuous in vps27Δ cells in which the prominent signal of Vps23-GFP on endosomes is eliminated, in a PalF- and alkaline pH-dependent manner. Dual-channel epifluorescence microscopy showed that PalC arrives at cortical complexes before PalA. As PalC recruitment is PalA independent and PalA recruitment is PalC dependent but PalB independent, these data complete the participation order of Pal proteins in the pathway and strongly support a model in which pH signalling takes place in ESCRT-containing, plasma-membrane-associated, rather than endosome-associated, complexes.
authors.affiliation: Department of Molecular Medicine, Centro de Investigaciones Biológicas CSIC, Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Galindo
authors.forename: Ana María
authors.initials: AM
authors.lastname: Calcagno-Pizarelli
authors.forename: Herbert N
authors.initials: HN
authors.lastname: Arst
authors.forename: Miguel Ángel
authors.initials: MÁ
authors.lastname: Peñalva
mesh_terms: D001233:Aspergillus nidulans / Q000166:cytology* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015398:Signal Transduction
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport