Spatial relationships in pH signalling in a model filamentous fungus: roles of MVB pathway and plasma membrane components

A vitally important feature of the environment of an organism is the degree of acidity or alkalinity, i.e. the pH. Fungi tailor the combinations of the enzymes they secrete, the nutrient transporters they synthesise and the products (such as toxins and antibiotics) they export to the pH of their environment. The ability of fungi to adapt to a range of environmental pH is crucial to their ability to infect both plants and animals (including humans) and to their ability to produce toxins such as aflatoxin and antibiotics such as penicillin and cephalosporin. Our work is directed towards determining how a signal is triggered by environmental alkalinity, ultimately leading to the processing of a DNA-binding protein such that it is able to 'turn on' fungal genes whose action is appropriate to alkaline environments and 'turn off' those appropriate to acidic environments. Previous work has shown that seven proteins are exclusively involved of which one is the DNA-binding protein and the other six are involved in transmitting the pH signal. These six signalling proteins include two located at the cell surface, one of which facilitates localisation of the other which is probably the pH sensor, a protein interacting with one of the surface proteins, two proteins interacting with a component of the cellular protein-sorting machinery of which one also interacts with the DNA-binding protein and a protein-cutting enzyme involved in activating the DNA-binding protein. In addition, such signalling of environmental pH involves some components of intracellular structures whose other job is the sorting of certain proteins towards an appropriate location within the cell or towards destruction. In this project we will use the model filamentous fungus Aspergillus nidulans, a relatively benign organism but related to the serious opportunistic pathogen Aspergillus fumigatus, the aflatoxin-producing agricultural scourge Aspergillus flavus and the filamentous fungus used in the fermentation to produce sake Aspergillus oryzae. We shall determine which of the protein sorting components are involved in signalling environmental pH and examine the consequences of mutations affecting those components. In some cases these mutations seriously impair growth of the fungus but other mutations can abrogate that impairment. We shall determine the mechanism of abrogation. We shall determine whether the demands of pH signalling result in a shortage of those components also needed for protein sorting and whether the sorting machinery is able to attenuate pH signalling. Finally we will investigate how the localisation of the pH sensor at the cell surface is assisted by the other pH signalling cell surface protein.

Work in yeasts and our background work has established involvement of multivesicular body (MVB) components in fungal ambient pH signal transduction. We will delete a number of genes encoding class E MVB components (vps) and determine their effects on pH signalling and on MVB function in the model filamentous fungus Aspergillus nidulans. Whilst deleting several vps genes we have obtained mutations (vsu) partially suppressing the severe growth defects resulting from vps deletions. We will characterise these vsu mutations and determine the basis of suppression. We will determine whether there is physiologically significant competition between complexes involved in pH signalling and those involved in cargo sorting/endosomal membrane inward invagination/ESCRT disassembly for MVB components common to both types of complex and whether transcriptional regulation of genes encoding these components is elevated under pH signalling (alkaline) conditions. We have identified selection techniques for obtaining vps mutations classically and will use these to obtain a mutant collection to further illuminate the roles of MVB components in pH signalling as well as helping to elucidate MVB function in this purely filamentous organism. We have also identified reversion techniques for obtaining further vps suppressors and will use these to increase the scope of the mutant collection. Our collaborators have identified a protein-protein interaction involving PalB, the cysteine protease responsible for the first, pH-dependent (activating) cleavage of the PacC transcription factor mediating pH regulation and we will determine whether this interaction physiologically down-regulates pH signalling. Finally we will investigate the mechanism by which the plasma membrane-localised pH signalling component PalI assists plasma membrane localisation of the putative pH sensor PalH through looking at their relative localisations and their possible interaction, including interactions via other proteins.