Toward the discovery of biological functions associated with the mechanosensor Mtl1p of Saccharomyces cerevisiae via integrative multi-OMICs analysis.

Functional analysis of the Mtl1 protein in Saccharomyces cerevisiae has revealed that this transmembrane sensor endows yeast cells with resistance to oxidative stress through a signaling mechanism called the cell wall integrity pathway (CWI). We observed upregulation of multiple heat shock proteins (HSPs), proteins associated with the formation of stress granules, and the phosphatase subunit of trehalose 6-phosphate synthase which suggests that mtl1Δ strains undergo intrinsic activation of a non-lethal heat stress response. Furthermore, quantitative global proteomic analysis conducted on TMT-labeled proteins combined with metabolome analysis revealed that mtl1Δ strains exhibit decreased levels of metabolites of carboxylic acid metabolism, decreased expression of anabolic enzymes and increased expression of catabolic enzymes involved in the metabolism of amino acids, with enhanced expression of mitochondrial respirasome proteins. These observations support the idea that Mtl1 protein controls the suppression of a non-lethal heat stress response under normal conditions while it plays an important role in metabolic regulatory mechanisms linked to TORC1 signaling that are required to maintain cellular homeostasis and optimal mitochondrial function.

Protein Interactions of the Mechanosensory Proteins Wsc2 and Wsc3 for Stress Resistance in Saccharomyces cerevisiae.

Antifungal drug discovery and design is very challenging because of the considerable similarities in genetic features and metabolic pathways between fungi and humans. However, cell wall composition represents a notable point of divergence. Therefore, a research strategy was designed to improve our understanding of the mechanisms for maintaining fungal cell wall integrity, and to identify potential targets for new drugs that modulate the underlying protein-protein interactions in Saccharomyces cerevisiae This study defines roles for Wsc2p and Wsc3p and their interacting protein partners in the cell wall integrity signaling and cell survival mechanisms that respond to treatments with fluconazole and hydrogen peroxide. By combined genetic and biochemical approaches, we report the discovery of 12 novel protein interactors of Wsc2p and Wsc3p Of these, Wsc2p interacting partners Gtt1p and Yck2p, have opposing roles in the resistance and sensitivity to fluconazole treatments respectively. The interaction of Wsc2p with Ras2p was confirmed by iMYTH and IP-MS approaches and is shown to play a dominant role in response to oxidative stress induced by hydrogen peroxide. Consistent with an earlier study, Ras2p was also identified as an interacting partner of Wsc1p and Mid2p cell wall integrity signaling proteins. Collectively, this study expands the interaction networks of the mechanosensory proteins of the Cell Wall Integrity pathway.

Identification and Functional Testing of Novel Interacting Protein Partners for the Stress Sensors Wsc1p and Mid2p of Saccharomyces cerevisiae.

Wsc1p and Mid2p are transmembrane signaling proteins of cell wall stress in the budding yeast Saccharomyces cerevisiae When an environmental stress compromises cell wall integrity, they activate a cell response through the Cell Wall Integrity (CWI) pathway. Studies have shown that the cytoplasmic domain of Wsc1p initiates the CWI signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor. Binding of Rom2p to the cytoplasmic tail of Wsc1p requires dephosphorylation of specific serine residues but the mechanism by which the sensor is dephosphorylated and how it subsequently interacts with Rom2p remains unclear. We hypothesize that Wsc1p and Mid2p must be physically associated with interacting proteins other than Rom2p that facilitate its interaction and regulate the activation of CWI pathway. To address this, a cDNA plasmid library of yeast proteins was expressed in bait strains bearing membrane yeast two-hybrid (MYTH) reporter modules of Wsc1p and Mid2p, and their interacting preys were recovered and sequenced. 14 previously unreported interactors were confirmed for Wsc1p and 29 for Mid2p The interactors' functionality were assessed by cell growth assays and CWI pathway activation by western blot analysis of Slt2p/Mpk1p phosphorylation in null mutants of each interactor under defined stress conditions. The susceptibility of these strains to different stresses were tested against antifungal agents and chemicals. This study reports important novel protein interactions of Wsc1p and Mid2p that are associated with the cellular response to oxidative stress induced by Hydrogen Peroxide and cell wall stress induced by Caspofungin.

Functional and genetic interactions of TOR in the budding yeast Saccharomyces cerevisiae with myosin type II-deficiency (myo1Δ).

BACKGROUND: Yeast has numerous mechanisms to survive stress. Deletion of myosin type II (myo1Δ) in Saccharomyces cerevisiae results in a cell that has defective cytokinesis. To survive this genetically induced stress, this budding yeast up regulates the PKC1 cell wall integrity pathway (CWIP). More recently, our work indicated that TOR, another stress signaling pathway, was down regulated in myo1Δ strains. Since negative signaling by TOR is known to regulate PKC1, our objectives in this study were to understand the cross-talk between the TOR and PKC1 signaling pathways and to determine if they share upstream regulators for mounting the stress response in myo1Δ strains. RESULTS: Here we proved that TORC1 signaling was down regulated in the myo1Δ strain. While a tor1Δ mutant strain had increased viability relative to myo1Δ, a combined myo1Δtor1Δ mutant strain showed significantly reduced cell viability. Synthetic rescue of the tor2-21(ts) lethal phenotype was observed in the myo1Δ strain in contrast to the chs2Δ strain, a chitin synthase II null mutant that also activates the PKC1 CWIP and exhibits cytokinesis defects very similar to myo1Δ, where the rescue effect was not observed. We observed two pools of Slt2p, the final Mitogen Activated Protein Kinase (MAPK) of the PKC1 CWIP; one pool that is up regulated by heat shock and one that is up regulated by the myo1Δ stress. The cell wall stress sensor WSC1 that activates PKC1 CWIP under other stress conditions was shown to act as a negative regulator of TORC1 in the myo1Δ mutant. Finally, the repression of TORC1 was inversely correlated with the activation of PKC1 in the myo1Δ strain. CONCLUSIONS: Regulated expression of TOR1 was important in the activation of the PKC1 CWIP in a myo1Δ strain and hence its survival. We found evidence that the PKC1 and TORC1 pathways share a common upstream regulator associated with the cell wall stress sensor WSC1. Surprisingly, essential TORC2 functions were not required in the myo1Δ strain. By understanding how yeast mounts a concerted stress response, one can further design pharmacological cocktails to undermine their ability to adapt and to survive.