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Functional and Biochemical Analysis of Interacting Protein Partners of Wsc1p and Mid2p Stress Sensors of Saccharomyces cerevisiae
Başlık:
Functional and Biochemical Analysis of Interacting Protein Partners of Wsc1p and Mid2p Stress Sensors of Saccharomyces cerevisiae
Yazar:
Santiago Cartagena, Ednalise, author.
ISBN:
9780438016156
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (239 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: José R. Rodríguez-Medina Committee members: Abel J. Baerga-Ortíz; Suranganie Dharmawardhane-Flanagan; Igor Stagljar; Carlos A. Torres-Ramos.
Özet:
The cell wall of the budding yeast Saccharomyces cerevisiae can be affected by environmental and genetic stresses that compromise cell wall integrity. Previous studies demonstrate that stresses such as antifungal drugs, oxidative chemical reagents, temperature, pH changes, deficiency of the myosin type II protein (Myo1p), among others can alter the cell wall composition of yeast cells and activate a mechanism to reestablish their cellular integrity. The Wsc protein family, Mid2p, and Mtl1p transmembrane signaling proteins are the sensors responsible for monitoring cell wall integrity and cellular stress. They activate this cellular response through the PKC1-dependent Cell Wall Integrity Pathway (CWIP). It has been demonstrated that the PKC1 CWIP is constitutively activated in myo1Delta cytokinesis mutants but the PKC1 CWIP sensor proteins responsible for detecting the cytokinesis stress signal provoked by MYO1 gene deletion remained unclear. Previous studies have shown that the cytoplasmic domain of Wsc1p initiates the PKC1 signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor (GEF). Binding of Rom2p to the cytoplasmic tail requires the dephosphorylation of specific serine residues but the mechanism by which the sensors are dephosphorylated and how they subsequently interact with Rom2p remains unclear. The first hypothesis for this study was that cell wall stress sensors can detect the stress signal caused by a deletion of the MYO1 gene and thereby activate the PKC1 CWIP. To test this hyphotesis, we constructed double deletion mutants combining sensor genes with and the MYO1 gene, and we assessed their viability and PKC1 CWIP activation status by performing Western blot analysis of Slt2p phosphorylation. The second hypothesis of this study was that Wsc1p and Mid2p are physically associated, either directly or indirectly with additional interacting proteins that facilitate Rom2p interaction and may regulate the activation of PKC1 signaling. To address this hyphotesis, 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 the interacting preys were recovered and sequenced. The interactors' functionality in PKC1 CWIP activation was assessed in null mutants of each interactor under specific conditions of stress and at defined temperatures. The susceptibility of these strains was further tested against different stressors, including several antifungal agents. The viability assays performed on myo1Delta strains confirmed that Wsc1 and Wsc3 sensor proteins are important for survival of myo1Delta strains. The low level of Slt2p phosphorylation in the wsc1Deltamyo1Delta and mid2Delta myo1Delta double mutants confirmed that Wsc1p and Mid2p are positive regulators of this pathway in myo1Delta strains and that cytokinetic stress is directly associated with cell wall stress in these strains. In wild type strains at 30°C, 14 novel interactors were confirmed for Wsc1p and 31 for Mid2p. A significant reduction in the number of interactors was observed when the MYTH screen was performed at 37°C. The Mid2p interactors Ras2p, Grx1p and Atx1p were required for efficient growth upon exposure to the oxidizing agent hydrogen peroxide, while Mid2p and Wsc1p was essential for normal growth in response to the antifungal drug Caspofungin. The interactions of Ras2p-Mid2p and Ras2p-Wsc1p were required for PKC1 CWI pathway activation in response to Caspofungin treatment. Overall, our study reports a novel function in the responses to Caspofungin cell wall stress and Hydrogen Peroxide oxidative stress that involves a shared interaction by Ras2p with Wsc1p and Mid2p. A subset of these specific protein-protein interactions are proposed as components of putative signaling complexes associated with oxidative and cell wall stress resistance. We conclude that these protein-protein interactions required for PKC1 CWIP regulation and cell survival are an attractive target for development of antifungal therapies.
Notlar:
School code: 0282
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
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