Identification and Functional Testing of Six Interactors of the Transmembrane Sensor Mtl1p in the Budding Yeast Saccharomyces cerevisiae

Abstract

Protein–protein interactions (PPIs) are often central to their cellular functions. These functions are often influenced by PPIs at all levels, including structural, energy metabolism, and signaling processes among others. The latter are crucial, as signaling functions often promote cell viability. We hypothesize that cytosolic protein interacting partners of the transmembrane sensor Mtl1p in Saccharomyces cerevisiae have a role in the stress response, promoting cell viability by modulation of the signaling process through the PKC1–dependent Cell Wall Integrity (CWI) Pathway.ObjectiveThe objective of our study is to identify cytosolic interactors of Mtl1p and to assess their relative importance in promoting cell viability upon exposure to diverse environmental stresses.MethodologyUsing membrane yeast two hybrid (MYTH) assays we have identified nine full–length proteins, and six other partial clones with C–terminals of various lengths that interact physically with the cytosolic domain of Mtl1p. In addition, using Western blots and growth curves we have made functional tests of deletion mutants corresponding to five of the identified full–length interactors, FPR1, ZEO1, RPL40a, EGD2, and CPR1, and one of the truncated interactors, RAS2, to assess their relative importance in promoting cell viability under oxidative the stress produced by exposure to H2O2, and cell wall stress produced by the antifungal Caspofungin.ResultsIn CSM broth cultures at 27°C, growth of the ras2Δ and zeo1Δ strains was severely impaired by 8 hours treatment with 0.5 mM H2O2, with a 50% reduction of cell concentration yields compared to the untreated wild type strain (WT) which is our reference standard. As determined by WB analyses, the ras2Δ strain treated with 1 mM H2O2 for two hours showed reduced levels of PKC1–Slt2p accumulation, indicative of CWI Pathway signaling, when compared to both the untreated ras2Δ and WT strains. Compared to the untreated WT, all three strains exhibit severely impaired growth when treated with 10 ng/ml Caspofungin at 27°C, especially the zeo1Δ strain. Moreover, all three strains show increased levels of PKC1–Slt2p signaling when treated with 75 ng/ml Caspofungin for two hours.ConclusionsOur results show that selective deletion of Mtl1p interactors like Ras2p or Zeo1p in S. cerevisiae are required to confer stress–specific susceptibility phenotypes. We propose that Ras2p modulates PKC1‐dependent Slt2p signaling on exposure to H2O2, perhaps directly by interacting with Mtl1p or indirectly by exerting an influence on other signaling pathways through a cross‐talk mechanism.Support or Funding InformationThis work is supported in part by NIH grants from NIGMS‐INBRE P20GM103475, NIGMSRISE R25GM061838, and NIMHD‐RCMI G12 MD007600.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.