Análisis genético-molecular del papel de la vía endocítica en la ruta de transducción de la señal de pH ambiental en Aspergillus nidulans

Abstract

[EN]: Regulation of gene expression by ambient pH in filamentous fungi and yeast is mediated by the pal/RIM signaling pathway and the transcription factor PacC/Rim101. The ambient pH signaling pathway is conserved in the fungal kingdom and has been mainly studied in the filamentous fungus Aspergillus nidulans and in the yeast Saccharomyces cerevisiae, although several studies have also been carried out in the yeasts Yarrowia lipolitica and Candida albicans. Ambient pH signal transduction involves the products of the pal genes and the transcription factor PacC in A. nidulans, and the corresponding RIM homologs in S. cerevisiae. The pal/RIM signaling pathway is activated in response to external pH increase. In response to this signal, the PacC transcription factor in A. nidulans and Rim101 in S. cerevisiae are activated by proteolytical processing. Studies carried out in A. nidulans showed that the proteolytical processing of the transcription factor regulates its nucleo-citoplasmic localization. The molecular mechanisms involved in the transduction of the ambient pH signal are highly conserved among fungi. The different components of the pal/PacC signaling pathway in A.nidulans have their corresponding homologs in the RIM pathway in S. cerevisiae. The putative ambient pHsensing complex contains a protein with 7 transmembrane domains (7TM), PalH in A. nidulans. In S. cerevisiae, two PalH homologs, Rim21 and Dfg16, have been reported, and both are required for pH signaling. Besides these 7TM proteins, there is a 3TM protein, which is also involved in pH signal reception: PalI in A. nidulans and its homolog Rim9 in S. cerevisiae. Other two main components of the signaling pathway, PalA and PalB in A. nidulans and their respective homologs Rim20 and Rim13 in S. cerevisiae, are likely involved in the proteolytical processing of the transcription factor. PalB/Rim13, which is predicted to be, by sequence analysis, a cysteine protease of the calpain family, appears to be responsible for the pH-dependent proteolytic processing of PacC/Rim101, whereas the proteasome would be responsible for a second proteolytical cleavage in PacC. PalA/Rim20 contains a Bro1 domain and interacts with the transcription factor PacC/Rim101. PalA recognizes two YPXL/I motifs in PacC, located at each side of the first proteolytical cleavage site, and the interaction of PalA with PacC is required for this first proteolytic processing step, presumably catalyzed by PalB. A second protein with a Bro1 domain, PalC in A. nidulans and its homolog Ygr122w in S. cerevisiae, appears to have a function in the signal transduction from the pH sensor to the complex involved in proteolytic processing of the transcription factor. Finally, our previous work in A. nidulans showed that

the last component of this pathway, PalF (Rim8 in S. cerevisiae), interacts with the C-terminal cytoplasmic tail of the 7TM protein PalH. Previous studies uncovered an unexpected link between ambient pH signaling and the endocytic pathway in fungi. PalA, and its yeast ortholog Rim20, were shown to interact in the two-hybrid system with Vps32 (Snf7), a component of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, which plays an essential role in multivesicular body (MVB) formation. MVB results from the maturation of the late endosome by invagination of vesicles that contain membrane proteins from Golgi or the plasma membrane, enroute to be processed or degraded in the vacuole. The fusion of the MVB membrane with that of the vacuole allows the discharge of these vesicles into the vacuole lumen and the proteolyses of its content. The formation of these vesicles by invagination of the endosome membrane requires a multi-protein complex, with several sub-complexes, ESCRT-I, ESCRT-II and ESCRT-III that associate to the endosome membrane. Snf7, a component of the ESCRT-III complex, recruits Bro1, a protein of the PalA/Rim20 family, which acts as an adaptor for the Doa4 deubiquitinase. A second possible link between ambient pH signaling and the

endocytic pathway is that PalF shares sequence similarities with mammalian arrestins. Arrestins were originally discovered by virtue of their ability to bind to activated 7TM receptors, leading to desensitization of these receptors. This regulatory mechanism uncouples receptors from heterotrimeric G proteins and inactivates the corresponding signaling pathway. It was discovered later on that these proteins play important additional functions and act as adaptors in several processes. On one hand, they are involved in the activation of alternative signaling pathways, such as MAP kinases pathway, by mediating the recruitment of components of these signaling pathways to the activated receptors. Most importantly, they play a key role in endocytosis of 7TM receptors since they work as adaptor between these receptors and the endocytic machinery. The internalization by endocytosis of activated 7TM receptors lead to the dephosphorylation, resensitization and recycling of these receptors to the plasma membrane or their traffic and degradation in the lysosome. Arrestins play a key role in this process by mediating the interaction between the activated and phosphorylated receptor and several components of the endocytic machinery including clathrin and clathrin adaptor AP2. The adaptor function of arrestins is based on their ability to interact with other proteins once bound to the 7TM receptor. Several studies indicate that the interaction with the activated and phosphorylated receptor produces a conformational change in the arrestin and allows the interaction of other proteins with inaccessible domains in its inactive conformation. However, this conformational change is not the only mechanism that regulates arrestin function in 7TM receptors endocytosis. It has also been demonstrated that phosphorylation / dephosphorylation and ubiquitination of arrestins play a key role in this process. For example, the beta-arrestin is constitutively phosphorylated in a serine residue closed to the clathrin binding site. Arrestin dephosphorylation in response to 7TM receptor activation is a requirement for clathrin binding and allows receptor endocytosis. Arrestin ubiquitination in response to 7TM receptor activation is another key regulatory mechanism involved in receptor endocytosis. PalF shares with mammal arrestins both sequence similarities and the ability to interact with the cytoplamic domains of 7TM proteins. We thus hypothesized that PalF could, as mammalian ß-arrestins, function as an endocytic adaptor, thus providing a second possible link between ambient pH signaling and the endocytic pathway.