Functional, genomic and molecular characterisation of Mtl1, an element of the CWI pathway of Saccharomyces cerevisiae with a role in the oxidative stress response

Laburpena

The eukaryotic microorganism Saccharomyces cerevisiae serves as a model system in which to study the signal transduction pathways involved in the oxidative stress response. Up to date, there is no evidence of any MAPK cascade which is specific to oxidative signals. Mtl1 is a member of the CWI pathway, which functions as a cell wall sensor for oxidative stress. In the present study, we propose an essential role for Mtl1 in signaling oxidative stress and quiescence to the CWI pathway and to the general stress response through the inhibition of either Tor1 or Ras2 functions. The Mtl1 function is required i) to induce ribosomal gene repression, ii) to induce the general stress response driven by the transcription factor Msn2/Msn4, and iii) to activate the CWI pathway in response to both oxidative stress and glucose starvation. The signaling from Mtl1 to Tor1 and/or Ras2 inhibition under these conditions occurs through Rom2 and Rho1, and probably through Pkc1, at least that signal which target is the ribosomal gene expression. We demonstrate that the Mtl1 cytoplasmic domain physically interacts with the GEF Rom2. Our data indicate that Slt2 activity is really essential in terms of cell survival in quiescent conditions. However, in response to oxidative stress the contribution of Msn2/Msn4 function is more significant. In addition, we demonstrate that deletion of either TOR1 or RAS2 is sufficient to activate Slt2 upon the above mentioned stress conditions, independently on Mtl1. These data suggested that CWI, TOR and Ras-cAMP provide diverse cross talks in order to assure the cells to appropriately adapt metabolism and growth. We demonstrate that Mtl1 is N-glycosylated and highly O-mannosylated mostly by Pmt2 mannosyltransferase. Mtl1 locates to the cell periphery, bud, septum, and to the tip of the shmoo. Mtl1 O-mannosylation confers its proper localization. We provide evidence for the importance of protein O-mannosylation in oxidative stress response, through at least Mtl1. This is the first report suggesting a role of protein Omannosylation in cell survival upon TOR blockage. Mtl1 O-mannosylation by Pmt2 is required to elicit cellular responses to TOR inhibition. Both Pmt2 and Mtl1 play positive roles in the chronological life span.