Department of Molecular Medicine, Centro de Investigaciones Biológicas CSIC, Madrid, Spain.
J Cell Sci. 2012 Apr 1;125(Pt 7):1784-95. doi: 10.1242/jcs.098897. Epub 2012 Feb 17.
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.
真菌 pal/RIM 信号通路通过调节基因表达来响应环境 pH 值，除了专门的蛋白外，还涉及到几个 ESCRT 复合物的成分，这表明 pH 值信号蛋白在内体平台上组装。在构巢曲霉中，专门的 Pal 蛋白包括质膜受体 PalH 和与其偶联的阻滞蛋白 PalF，在碱性 pH 条件下，PalF 被泛素化，以及三个潜在的内体 ESCRT-III 相关蛋白，包括 Vps32 相互作用蛋白 PalA 和 PalC 以及 Vps24 相互作用蛋白 calpain 样 PalB。我们通过将环境 pH 值升高到碱性条件来激活途径，研究了信号转导发生的亚细胞位置。Vps32 相互作用蛋白 PalA 和 PalC 并没有定位在内体上，而是在碱性诱导的皮层结构中短暂地共定位，这依赖于 PalH、Vps23 和 Vps32，但不依赖于 Vps27。当 Vps4 缺失时，这些皮层结构更加稳定，表明它们的半衰期依赖于 ESCRT-III 的解体。下拉研究表明，Vps23 与 PalF 强烈相互作用，但共免疫沉淀物仅来自提取物中泛素化的 PalF 形式。我们证明，在生理水平表达的 Vps23-GFP 也被招募到皮层结构中，在 vps27Δ 细胞中，Vps23-GFP 在内涵体上的显著信号被消除，这是一种依赖于 PalF 和碱性 pH 值的方式。双通道荧光显微镜显示，PalC 比 PalA 先到达皮层复合物。由于 PalC 的募集不依赖于 PalA，而 PalA 的募集依赖于 PalC 但不依赖于 PalB，这些数据完整了 Pal 蛋白在该途径中的参与顺序，并强烈支持了 pH 值信号发生在含有 ESCRT 的、质膜相关的、而不是内涵体相关的复合物中的模型。
