Receptor-independent Ambient pH Signaling by Ubiquitin Attachment to Fungal Arrestin-like PalF^*

América Hervás-Aguilar, Antonio Galindo, , and Miguel A. Peñalva¹

From the Department of Molecular and Cellular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain

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Abstract: 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.

Key Words: Fungi • 7-Helix Receptor • Plasma Membrane • Signal Transduction • Ubiquitination • Arrestin • ESCRT

Received for publication February 15, 2010. Revision received March 22, 2010.

¹ To whom correspondence should be addressed: Ramiro de Maeztu 9, 28040 Madrid, Spain. Tel.: 34-91-837-3112 (ext. 4358); Fax: 34-91-536-0432; E-mail: penalva{at}cib.csic.es.

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