S. cerevisiae putative ammonium exporters Ato are localised in lipid rafts.
Marketa Ricicova, Zdena Palkova
Genetics and Microbiology, Charles University, Vinicna 5, Prague 2, 128 44, Czech Republic
Ammonia functions in yeast colonies as a long range signal which is important for their development and adaptation to depletion of nutrients. Recently, we have discovered three new membrane proteins Ato1p, Ato2p and Ato3p (Ammonium Transport Outwards) that seems to be involved in ammonium export during ammonia production in S. cerevisiae colonies. All three ATO genes are strongly and quickly induced just at the moment when colonies start to produce ammonia. Deletion of each of the ATO genes leads to severe decrease of ammonia production. Ato protein sequence analysis predicted their localisation in the plasma membrane and it revealed Ato1p region exhibiting homology with „ammonium transporter signature“of the C. elegans putative ammonium transporter. We described 20 other proteins homologous to Ato proteins occurring predominantly in yeast and bacteria species (Palkova et al. 2002, Mol Bio Cell 13: 3901-3914). Expression of ATO1 -GFP, ATO2 -GFP and ATO3 -GFP confirmed localisation of Ato proteins in the plasma membrane. In accord with previous findings, Ato-GFP fluorescence starts to be visible at the beginning of ammonia production. Confocal microscopy indicates the association of Ato proteins with lipid raft domains. This prediction was confirmed by lipid raft isolation and detection of all three fusion proteins in the detergent-resistant membrane fraction. This work was supported by grant 204/05/0294.