The fungal pal/RIM signalling pathway regulates gene expression in response to environmental pH. In Aspergillus nidulans it involves six dedicated proteins, PalA, PalB, PalC, PalF, PalH and PalI, which mediate the proteolytic activation of the transcription factor PacC. In addition, it requires several components of the ESCRT (endosomal sorting complex required for transport) complexes, which mediate multivesicular body biogenesis at endosomes. This fact suggested that pH signalling proteins might assemble on endosomal platforms. Amongst Pal proteins are the plasma membrane receptor PalH and its coupled arrestin, PalF. PalF becomes ubiquitylated in an alkaline pH8208;Â and PalH8208;dependent manner; three other Pal proteins are ESCRT8208;III associates, and thus they were considered potentially endosomal. These are the Vps328208;interactors PalA and PalC and the Vps248208;interactor calpain8208;like PalB. Therefore previous models speculated that intracellular traffic would mediate the connection between plasma membrane8208;Â and endosomal membrane8208;associated complexes. We studied by in vivo microscopy the subcellular localization 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 oscillate at the plasma membrane, transiently co8208;localising to alkaline pH8208;induced cortical structures in a PalH8208; dependent manner. Notably, the assembly of this cortical structures is Vps23 (ESCRT8208;I)8208;Â and Vps32 (ESCRT8208;III)8208; dependent but Vps27 (ESCRT8208;0)8208;independent. These cortical structures are dramatically more stable under conditions leading to Vps4 deficiency, indicating that their half8208;life depends on ESCRT8208;III disassembly. Pull8208;down studies demonstrated that Vps23 interacts strongly with the PalF arrestin. Notably Vps23 co8208;immunoprecipitates exclusively ubiquitylated PalF forms from extracts. Endogenously tagged Vps238208;GFP is also recruited to cortical structures, in addition to endosomes, in a PalF8208;Â and alkaline pH8208;dependent manner. These Vps238208;GFP structures become particularly obvious in vps27916; cells where the conspicuous endosomal localisation of Vps23 is prevented. Dual8208;channel time8208;lapse epifluorescence microscopy showed that PalC arrives to cortical complexes before PalA. As PalC recruitment is PalA8208;independent and PalA recruitment is PalC8208;dependent but PalB8208;independent, these data complete the participation order of Pal proteins in the pathway. Importantly, they strongly support a model in which pH signalling takes place in ESCRT8208;containing, plasma membrane8208;associated, rather than endosome8208; associated, signalling complexes.
Full conference title:
11 th European Conference on Fungal Genetics
- ECFG 11th (2012)