Department of Molecular and Cellular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain.
J Biol Chem. 2010 Jun 4;285(23):18095-102. doi: 10.1074/jbc.M110.114371. Epub 2010 Apr 5.
The seven-transmembrane receptor PalH and its coupled, positive-acting arrestin-like protein PalF are key components of a molecular sensor that in Aspergillus nidulans and other ascomycete fungi mediates activation of an intracellular signaling cascade by alkaline ambient pH. PalF is ubiquitinated in an alkaline pH- and PalH-dependent manner. We show here that PalF assists the plasma membrane localization of PalH and that PalF overexpression slightly hypersensitizes the pathway to alkaline pH but does not bypass the need for the ambient pH signal receptor in signaling. In contrast, covalent attachment of Ub to PalF activates the signaling pathway under acidic pH conditions in which the pathway is normally inactive, demonstrating a positive role for ubiquitination. We further show that PalF acts upstream of, or in concert with, the Bro1 domain-containing pH signaling protein PalC, which is normally recruited to cortical structures likely to represent active pH signaling foci under neutral/alkaline pH conditions. In agreement with its pathway-activating consequences, expression of PalF-Ub also promotes PalC cortical recruitment under acidic conditions. Notably, our data establish that expression of PalF-Ub, at approximately physiological levels, in a null palH background leads to a considerable degree of signaling even in the complete absence of the receptor. Thus PalF ubiquitination is a key, perhaps the sole, molecular trigger required for transmitting the alkaline pH signal to the downstream elements of the pathway.
七跨膜受体 PalH 及其偶联的正向作用的 arrestin 样蛋白 PalF 是一种分子传感器的关键组成部分，该传感器在 Aspergillus nidulans 和其他子囊菌真菌中介导细胞内信号级联反应的激活，该信号级联反应由碱性环境 pH 值触发。PalF 在碱性 pH 值和 PalH 依赖性方式下被泛素化。我们在这里表明，PalF 协助 PalH 的质膜定位，并且 PalF 的过表达略微增加了该途径对碱性 pH 值的敏感性，但不会绕过信号通路中对环境 pH 值信号受体的需求。相比之下，Ub 与 PalF 的共价结合在酸性 pH 值条件下激活信号通路，而在正常情况下该途径是不活跃的，这证明泛素化具有积极作用。我们进一步表明，PalF 在上游或与 Bro1 结构域包含的 pH 信号蛋白 PalC 协同作用，PalC 通常被募集到皮质结构中，在中性/碱性 pH 值条件下可能代表活跃的 pH 信号焦点。与它的途径激活作用一致，PalF-Ub 的表达也促进了 PalC 在酸性条件下的皮质募集。值得注意的是，我们的数据表明，在 palH 缺失背景下，以大约生理水平表达 PalF-Ub 甚至在完全缺乏受体的情况下也会导致相当程度的信号传递。因此，PalF 泛素化是将碱性 pH 值信号传递给途径下游元件的关键（也许是唯一）分子触发因素。
